Pyruvate derivatives

ABSTRACT

Certain known and novel pyruvate derivatives are particularly active in restoring or preserving metabolic integrity in oxidatively competent cells that have been subjected to oxygen deprivation. These pyruvate-derived compounds include, but are not limited to oximes, amides, pyruvate analogues, modified pyruvate analogues, esters of pyruvate (e.g., polyol-pyruvate esters, pyruvate thioesters, glycerol-pyruvate esters and dihydroxyacetone-pyruvate esters). Such pyruvate derivatives (including single tautomers, single stereoisomers and mixtures of tautomers and/or stereoisomers, and the pharmaceutically acceptable salts thereof) are useful in the manufacture of pharmaceutical compositions for treating a number of conditions characterized by oxidative stress.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority as a continuation-in-part ofco-pending provisional applications: U.S. Serial No. 60/288,649, filedMay 3, 2001; U.S. Serial No. 60/295,314, filed Jun. 1, 2001; and U.S.Serial No. 60/368,456, filed Mar. 27, 2002, each incorporated herein byreference in its entirety. This application is also related toco-pending application U.S. Serial No. ______, filed on even dateherewith and entitled “PROCESS FOR SOLID SUPPORTED SYNTHESIS OFPYRUVATE-DERIVED COMPOUNDS”, incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

[0002] The present invention relates to pyruvate derivatives,particularly to derivatives having cytoprotective activity, andspecifically to a series of aromatic and peptidyl pyruvates. Theinvention is also directed to formulations and methods for treatingstroke, myocardial infarction and chronic heart failure, as well asother oxidative stress and/or inflammation related conditions that aretypically responsive to cellular enzyme modulation.

BACKGROUND INFORMATION

[0003] Pyruvate is a three-carbon (triose) ketoacid that is produced inbiological systems in the end stages of glycolysis, a product of sugarmetabolism. It is also a breakdown product of certain amino acids(alanine, glycine, cysteine, serine). Pyruvate can be reduced to lactatein the cytoplasm, a fermentative event in mammalian cells, oroxidatively decarboxylated to acetyl CoA in the mitochondrion.

[0004] There are known in the art a number of pyruvate derivatives. Ithas been suggested that pyruvate and certain pyruvate derivatives mayhave utility in treating certain disorders and promoting health. Forexample, pyruvate is sold as a dietary supplement for use in promotingweight loss and enhancing energy. It has also been suggested as atherapeutic intervention for clinical management of myocardialinsufficiency (Mallet, R. T., 2000, Proc. Soc. Exp. Biol. Med. 223(2):136-148) and to prevent the adverse effects of myocardial ischemia (U.S.Pat. No. 5,294,641). U.S. Pat. Nos. 5,075,210 and 4,988,245 describe theuse of pyruvate or pyruvate salts as a component in a cardioplegicsolution and in preservation solutions (perfusion fluids) for heartallografts before transplantation. U.S. Pat. No. 5,395,822 describes theuse of certain pyruvate salts to protect against neuronal degenerationas a consequence of ischemia.

[0005] U.S. Pat. No. 6,086,789 describes certain pyruvate derivatives asuseful for dermatologic indications as well as for treating diabeticketosis, myocardial ischemia, injured organs and hypercholesterolemia.Specifically, it ascribes these activities to various esters ofpyruvate, including polyol-pyruvate esters, pyruvate thioesters,glycerol-pyruvate esters, and dihydroxyacetone-pyruvate ester. RelatedU.S. Pat. No. 5,968,727 describes the use of pyruvate thiolesters, suchas cysteine, methionine and homocysteine, and glycerol pyruvate estersand dihydroxyacetone-pyruvate esters, in organ preservation solutionsand for treating ischemia. Similarly, certain pyruvate and pyruvyl aminoacid conjugates have been suggested for use in diabetes (e.g., U.S. Pat.Nos. 5,047,427 and 5,256,697).

[0006] It has, however, remained desired to provide new therapies forconditions characterized by oxidative stress, and particularly, forproviding neuroprotection in the event of cerebral ischemia; especiallydesired are agents that are effective even if first administered after asignificant period of time (e.g., about 5 or more hours) following anischemic insult.

SUMMARY OF THE INVENTION

[0007] The present invention is concerned with certain known and novelpyruvate derivatives that are particularly active in restoring orpreserving metabolic integrity in oxidatively competent cells that havebeen subjected to oxygen deprivation. These pyruvate-derived compoundsinclude, e.g., but are not limited to oximes, amides, pyruvateanalogues, modified pyruvate analogues, esters of pyruvate (e.g.,polyol-pyruvate esters, pyruvate thioesters, glycerol-pyruvate estersand dihydroxyacetone-pyruvate esters). Such pyruvate derivatives areuseful in the manufacture of pharmaceutical compositions for treating anumber of conditions characterized by oxidative stress, andparticularly, in providing neuroprotection in the event of cerebralischemia, even when administered a significant time interval after anischemic insult. In particular, the compositions of the presentinvention are useful in the treatment of stroke, as demonstrated byproviding neuroprotection in a standard experimental model of focalcerebral ischemia. They are also useful in the treatment of myocardialischemia (myocardial infarction), as well as other indicationscharacterized by oxidative stress and/or inflammation, including, butnot limited to, neurodegenerative disorders such as Alzheimer's,dementia, and Parkinson's disease; diabetes; renal disease; premenstrualsyndrome; asthma, cardiopulmonary inflammatory disorders; chronic heartfailure; rheumatoid arthritis; muscle fatigue; intermittentclaudication; and for the preservation of allograft tissue fortransplantation.

[0008] One aspect of the present invention concerns methods of treatmentand the manufacture of medicaments therefor, employing the compoundsrepresented by Formula I:

[0009] wherein:

[0010] the dashed line is a double bond one of the indicated positionsand a single bond in the other, or (where W is —N(OH)—C(O)—R^(d)) is asingle bond in both positions;

[0011] A is: optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl, optionally substituted heteroaralkyl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkyl, an optionally substituted nucleoside, an optionallysubstituted amino acid, an optionally substituted di-, tri- ortetra-peptide, —CH₂—C(O)—C(O)—O—R′ or —CH═C(OH)—C(O)—O—R′;

[0012] X is: —N(R′)—, —S—, —S(O)—, —S(O)₂—, —S—Y—S—, or a covalent bondto the sulfur atom of Cys or to the nitrogen atom of optionallysubstituted heterocyclyl;

[0013] Y is: optionally substituted aryl, optionally substitutedheteroaryl, an optionally substituted nucleoside, an optionallysubstituted amino acid, or an optionally substituted di-, tri- ortetra-peptide;

[0014] W is: ═O, ═N—OR^(a), ═N—NR^(b)R^(c), or N(OH)—R^(d);

[0015] Z is: —OR, —SR, or —NR^(b)R^(c);

[0016] R′ is: independently selected from hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl or optionallysubstituted aryl;

[0017] R is: hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl, optionallysubstituted heteroaralkyl, optionally substituted heterocyclyl oroptionally substituted heterocycloalkyl;

[0018] R^(a) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, or alkenyl;

[0019] R^(b) is: independently selected from hydrogen, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedaralkyl or optionally substituted cycloalkyl;

[0020] R^(c) is: independently selected from hydrogen or optionallysubstituted alkyl; and

[0021] R^(d) is: hydrogen, acyl or optionally substituted alkyl; or

[0022] R^(b) and R^(c) together with the nitrogen to which they areattached may form a 5- to 7-membered ring, optionally incorporating oneor two additional ring heteroatoms chosen from N, S, or O, and said ringbeing optionally substituted with one or more substituents independentlyselected from the group consisting of ═O, ═S, acyl, optionallysubstituted alkenyl, optionally substituted alkyl, (optionallysubstituted alkoxy)carbonyl, and (optionally substituted amino)carbonyl;

[0023] including single tautomers, single stereoisomers and mixtures oftautomers and/or stereoisomers, and the pharmaceutically acceptablesalts thereof, provided that where X is —S—, W is ═O, and Z is OH, A isnot 6-amino-3,5-dicyano-4-(optionally substituted phenyl)-pyridin-2-yl.

[0024] Another aspect of the present invention concerns the compoundsrepresented by Formula Ia:

[0025] where:

[0026] A is: substituted alkyl selected from: —CH₂—CH(OH)—CH₂—OH,—CH(CH₃)—CH(OH)—CH₂—OH, —CH(CH₃)—C(O)—N(H)—CH₂—COOH,—CH(CH₃)—C(O)—N(H)—CH₂—C(O)—O—CH₂—CH₃, —CH₂—C(O)—N(H)—CH₂—COOH,—CH₂—CH₂—C(O)—N(H)—CH₂—COOH, —CH(CH₃)—CH₂—C(O)—N(H)—CH₂—COOH, and—CH₂—CH(CH₃)—C(O)—N(H)—CH₂—COOH,

[0027] substituted heteroaryl selected from:5-chloro-1H-benzoimidazol-2-yl, 5-methoxy-1H-benzoimidazol-2-yl,4-oxo-3,4-dihydro-quinazolin-2-yl, benzoselenazol-2-yl, and5-substituted-benzothiazol-2-yl;

[0028] heterocyclyl selected from: thiazol, 2-thioxo-imidazolidin-1-yland morpholino, an optionally substituted nucleoside, or an optionallysubstituted di-, tri- or tetra-peptide, or

[0029] —CH₂—C(O)—C(O)—O—R′ or —CH═C(OH)—C(O)—O—R′;

[0030] R is: hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl, optionallysubstituted heteroaralkyl, optionally substituted heterocyclyl oroptionally substituted heterocycloalkyl;

[0031] R′ is: independently selected from hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl or optionallysubstituted aryl;

[0032] X is: —N(R′)—, —S—, —S(O)—, —S(O)₂—, —S—Y—S—, or a covalent bondto the sulfur atom of Cys or to the nitrogen atom of optionallysubstituted heterocyclyl;

[0033] Y is: optionally substituted aryl, optionally substitutedheteroaryl, an optionally substituted nucleoside, an optionallysubstituted amino acid, or an optionally substituted di-, tri- ortetra-peptide; and

[0034] Z is: —OR or —SR;

[0035] including single tautomers, single stereoisomers and mixtures oftautomers and/or stereoisomers, and the pharmaceutically acceptablesalts thereof. The invention also pertains to methods of treatment,pharmaceutical formulations and the manufacture of medicaments employingthe compounds of Formula Ia.

[0036] Another aspect of the present invention concerns the compoundsrepresented by Formula Ib:

[0037] where:

[0038] A is: optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl, optionally substituted heteroaralkyl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkyl, an optionally substituted nucleoside, an optionallysubstituted amino acid, an optionally substituted di-, tri- ortetra-peptide, —CH₂—C(O)—C(O)—O—R′ or —CH═C(OH)—C(O)—O—R′;

[0039] R is: hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl, optionallysubstituted heteroaralkyl, optionally substituted heterocyclyl oroptionally substituted heterocycloalkyl;

[0040] R^(a) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, or alkenyl;

[0041] R′ is: independently selected from hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl or optionallysubstituted aryl;

[0042] X is: —S—, —S(O)—, —S—Y—S—, or a covalent bond to the sulfur atomof Cys or to the nitrogen atom of optionally substituted heterocyclyl;

[0043] Y is: optionally substituted aryl, optionally substitutedheteroaryl, an optionally substituted nucleoside, an optionallysubstituted amino acid, or an optionally substituted di-, tri- ortetra-peptide; and

[0044] Z is: —OR or —SR;

[0045] including single tautomers, single stereoisomers and mixtures oftautomers and/or stereoisomers, and the pharmaceutically acceptablesalts thereof; provided that:

[0046] where X is —S—, A is not optionally substituted alkyl, benzyl oran N-arylpyrroline-2,5-dione-substituted phenyl,

[0047] and further provided that the compound of Formula 1b is not:

[0048] 2-hydroxyimino-3-p-tolylsulfanyl-propionic acid ethyl ester.

[0049] The invention also pertains to methods of treatment,pharmaceutical formulations and the manufacture of medicaments employingthe compounds of Formula Ib.

[0050] Another aspect of the present invention concerns the compoundsrepresented by Formula Ic:

[0051] where:

[0052] A is: optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl, optionally substituted heteroaralkyl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkyl, an optionally substituted nucleoside, an optionallysubstituted amino acid, an optionally substituted di-, tri- ortetra-peptide, CH₂—C(O)—C(O)—O—R′ or —CH═C(OH)—C(O)—O—R′;

[0053] W is: ═O, ═N—OR^(a), or —N(OH)—R^(d);

[0054] X is: —S—, —S(O)—, —S(O)₂—, —S—Y—S—, or a covalent bond to thesulfur atom of Cys or to the nitrogen atom of optionally substitutedheterocyclyl;

[0055] Y is: optionally substituted aryl, optionally substitutedheteroaryl, an optionally substituted nucleoside, an optionallysubstituted amino acid, or an optionally substituted di-, tri- ortetra-peptide;

[0056] R′ is: independently selected from hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl or optionallysubstituted aryl;

[0057] R^(a) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, or alkenyl;

[0058] R^(b) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl or optionallysubstituted cycloalkyl;

[0059] R^(c) is: hydrogen or optionally substituted alkyl; and

[0060] R^(d) is: hydrogen, acyl or optionally substituted alkyl; orR^(b) and R^(c) together with the nitrogen to which they are attachedmay form a 5- to 7-membered ring, optionally incorporating one or twoadditional ring heteroatoms chosen from N, S, or O, and said ring beingoptionally substituted with one or more substituents independentlyselected from the group consisting of ═O, ═S, acyl, optionallysubstituted alkenyl, optionally substituted alkyl, (optionallysubstituted alkoxy)carbonyl, and (optionally substituted amino)carbonyl;

[0061] including single tautomers, single stereoisomers and mixtures oftautomers and/or stereoisomers, and the pharmaceutically acceptablesalts thereof, provided that:

[0062] where X is —S—, A is not optionally substituted methyl,optionally substituted ethyl, optionally substituted benzyl, ortriphenylmethyl, and

[0063] where W is ═N—OR^(a) and X is a covalent bond to the sulfur atomof Cys, A is an optionally substituted di-, tri- or tetra-peptide.

[0064] The invention also pertains to methods of treatment,pharmaceutical formulations and the manufacture of medicaments employingthe compounds of Formula Ic.

[0065] In a preferred embodiment of Formulae I, Ia, Ib and Ic, where Ais a natural or substituted amino acid or peptide, A is selected fromthe group: Ala, Asn, Asp, Cys, Gln, Glu, Gly, Lys, Met, Ser and Thr,especially Ala, Asp, Cys, Glu and Gly. Further preferred are thosecompounds where A is a natural or substituted di- or tri-peptide,especially natural peptides and most preferably the tri-peptideGlu-Cys-Gly.

[0066] In another such preferred embodiment, A is an optionallysubstituted heteroaryl group, especially a nitrogen-containingoptionally substituted heteroaryl, and particularly where A is selectedfrom the group: imidazole, triazole, thiadiazole, oxadiazole,benzoselenazol, benzoimidazole and benzothiazole.

[0067] Further preferred in each of the foregoing embodiments are thosecompounds where X is —S— or a covalent bond.

[0068] Another preferred embodiment of the invention concerns thecompounds, represented by Formula II:

[0069] where:

[0070] R¹ is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, —C(O)—O—R′, —CH₂—SH, —CH₂—S—CH₂—C(W)—C(O)—Z,—CH₂—S—CH═C(OH)—C(O)—Z, —CH₂—S(O)—CH₂—C(W)—C(O)—Z, or—CH₂—S(O)—CH═C(OH)—C(O)—Z′;

[0071] R² is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedacyl;

[0072] R³ is: independently selected from hydrogen, optionallysubstituted lower alkyl, optionally substituted aralkyl, —CH₂—SH,—CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z;

[0073] R⁴ is: hydrogen, optionally substituted lower alkyl, optionallysubstituted aralkyl, optionally substituted heteroararalkyl, —CH₂—SH,—CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z;

[0074] R⁵ is: hydrogen, optionally substituted alkyl, or optionallysubstituted aryl;

[0075] R′ is: independently selected from hydrogen, optionallysubstituted alkyl, or optionally substituted aryl;

[0076] W is: ═O, ═N—OR^(a), ═N—NR^(b)R^(c); or —N(OH)—R^(d)

[0077] Z is: —OR, —SR, or —NR^(b)R^(c);

[0078] R is: hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl, optionallysubstituted heteroaralkyl, optionally substituted heterocyclyl oroptionally substituted heterocycloalkyl;

[0079] R^(a) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, or alkenyl;

[0080] R^(b) is: independently selected from hydrogen, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedaralkyl or optionally substituted cycloalkyl,

[0081] R^(c) is: independently selected from hydrogen or optionallysubstituted alkyl; and

[0082] R^(d) is: hydrogen, acyl or optionally substituted alkyl; or

[0083] R^(b) and R^(c) together with the nitrogen to which they areattached may form an 5- to 7-membered ring, optionally incorporating oneor two additional ring heteroatoms chosen from N, S, or O, and said ringbeing optionally substituted with one or more substituents independentlyselected from the group consisting of ═O, ═S, acyl, optionallysubstituted alkenyl, optionally substituted alkyl, (optionallysubstituted alkoxy)carbonyl, and (optionally substituted amino)carbonyl;

[0084] k is: 0, 1 or 2;

[0085] m is: 0, 1 or 2; and

[0086] n is: 0, 1, 2 or 3;

[0087] including single tautomers, single stereoisomers and mixtures oftautomers and/or stereoisomers, and the pharmaceutically acceptablesalts thereof, provided that at least one of R¹, R³ or R⁴ is—CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z. The inventionalso pertains to methods of treatment, pharmaceutical formulations andthe manufacture of medicaments employing the compounds of Formula II.

[0088] Of the compounds according to Formula II, preferred are thosecompounds the substituents of which are selected from the followinggroups:

[0089] R¹ is: —C(O)—O—R′ where R′ is hydrogen or lower alkyl;

[0090] R² is: hydrogen;

[0091] R³ is: —CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z

[0092] R⁴ is: hydrogen;

[0093] R⁵ is hydrogen or lower alkyl;

[0094] W is: ═O or ═N—OR′;

[0095] Z is: —OR or —NR^(b)R^(c);

[0096] R is: hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, or optionallysubstituted aralkyl;

[0097] R^(a) is: hydrogen or alkyl;

[0098] R^(b) is: C₁ to C₄ alkyl, phenyl or benzyl;

[0099] R^(c) is: hydrogen or C₁ to C₄ alkyl, or

[0100] R^(b) and R^(c) together with the nitrogen to which they areattached form a 6-membered ring selected from 4-optionallysubstituted-piperidin-1-yl and morpholin-4-yl; and

[0101] k, m and n are respectively: 0,2,1; 1,0,1; or 2,0,1.

[0102] Another aspect of the invention entails compounds made from thecompounds of Formula Ia, for example, where A is cysteine, bycyclization to give a dihydrothiazine-3,5-dicarboxylic acid or likederivative, as represented by Formula III:

[0103] where:

[0104] the dashed line represents a double bond at one or the other ofthe indicated positions, corresponding to a 3,4-dihydro-2H-[1,4]thiazineor a 3,6-dihydro-2H-[1,4]thiazine (or 5,6-dihydro-2H-[1,4]thiazine inthe nomenclature of the compounds where R^(3.5) is H);

[0105] R^(3.1) is: H where Formula III is a3,4-dihydro-2H-[1,4]thiazine, and is absent where Formula III is a3,6-dihydro-2H-[1,4]thiazine;

[0106] R^(3.2) is: H or C₁ to C₄ alkyl;

[0107] R^(3.3) and R^(3.4) are both H or are both C₁ to C₄ alkyl; and

[0108] R^(3.5) is: H, COOH, or —C(O)O—C₁ to C₄ alkyl;

[0109] including single tautomers, single stereoisomers and mixtures oftautomers and/or stereoisomers, and the pharmaceutically acceptablesalts thereof. The invention also pertains to methods of treatment,pharmaceutical formulations and the manufacture of medicaments employingthe compounds of Formula III.

[0110] In another aspect, the invention relates to a pharmaceuticalcomposition containing a therapeutically effective amount of a compoundof Formulae I, Ia, Ib, Ic, II or III or a pharmaceutically acceptablesalt thereof admixed with at least one pharmaceutically acceptableexcipient. Particularly preferred are those pharmaceutical compositionswherein the compound of Formulae I, Ia, Ib, Ic, II or III is selectedfrom the herein-described preferred embodiments.

[0111] In still another aspect, the invention relates to a method oftreatment by administering to a mammal in need of such treatment atherapeutically effective amount of a compound of Formulae I, Ia, Ib,Ic, II or III or a pharmaceutically acceptable salt thereof, where thedisease, condition or indication for which treatment is provided is:ischemia including stroke, cerebral ischemia, retinal ischemia,myocardial ischemia, myocardial infarction and post-surgical cognitivedysfunction; neurodegenerative disorders including Alzheimer's, dementiaand Parkinson's disease; peripheral neuropathy, including spinal cordinjury, head injury and surgical trauma; inflammatory disordersincluding diabetes, renal disease, premenstrual syndrome, asthma,cardiopulmonary inflammatory disorders, heart failure, rheumatoidarthritis, osteoarthritis, muscle fatigue and intermittent claudication;and for the preservation of allograft tissue and organs fortransplantation. Particularly preferred are those methods of treatmentand uses in the manufacture of pharmaceutical compositions therefore,wherein the compound of Formulae I, Ia, Ib, Ic, II or III is selectedfrom the herein-described preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

[0112] Definitions

[0113] As used in the present specification, the following words andphrases are generally intended to have the meanings as set forth below,except to the extent that the context in which they are used indicatesotherwise.

[0114] The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined below. Itwill be understood by those skilled in the art with respect to any groupcontaining one or more substituents that such groups are not intended tointroduce any substitution or substitution patterns (e.g., substitutedalkyl includes optionally substituted cycloalkyl groups, which in turnare defined as including optionally substituted alkyl groups,potentially ad infinitum) that are sterically impractical and/orsynthetically non-feasible.

[0115] Certain compound, reactant, or reaction parameter abbreviationsare defined as follows:

[0116] “DCM” refers to dichloromethane or methylene chloride

[0117] “DIC” refers to N,N-diisopropylcarbodiimide.

[0118] “DIPEA” refers to diisopropyl ethylamine.

[0119] “DMAP” refers to 4-N,N-dimethylamino pyridine.

[0120] “DMF” refers to N,N-dimethyl formamide.

[0121] “DTT” refers to dithiothreitol.

[0122] “EDT” refers to ethanedithiol.

[0123] “Eq.” refers to equivalent.

[0124] “Fmoc” refers to 9-fluorenylmethoxycarbonyl.

[0125] “GlyOH” refers to glycine.

[0126] “HOBt” refers to N-hydroxybenzotriazole.

[0127] “MeOH” refers to methanol.

[0128] “t-Bu” refers to t-butyl.

[0129] “TBTU” refers to2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate.

[0130] “TIS” refers to triisopropylsilane.

[0131] “TFA” refers to trifluoroacetic acid.

[0132] The term “acyl” refers to the groups —C(O)—H, —C(O)-(optionallysubstituted alkyl), —C(O)-(optionally substituted cycloalkyl),—C(O)-(optionally substituted alkenyl), —C(O)-(optionally substitutedcycloalkenyl), —C(O)-(optionally substituted aryl), —C(O)-(optionallysubstituted aralkyl), —C(O)-(optionally substituted heteroaryl),—C(O)-(optionally substituted heteroaralkyl), —C(O)-(optionallysubstituted heterocyclyl) and —C(O)-(optionally substitutedheterocycloalkyl).

[0133] The term “acyloxy” refers to the moiety —O-acyl, including, forexample, —O—C(O)-alkyl.

[0134] The term “alkenyl” refers to the monoradical branched orunbranched, unsaturated or polyunsaturated hydrocarbon chain, havingfrom about 2 to 20 carbon atoms, more preferably about 2 to 10 carbonatoms. This term is exemplified by groups such as ethenyl, but-2-enyl,and the like.

[0135] The term “alkoxy” refers to the groups —O-alkyl, —O-alkenyl,—O-cycloalkyl, —O-cycloalkenyl, and —O-alkynyl. Preferred alkoxy groupsare —O-alkyl and include, by way of example, methoxy, ethoxy, n-propoxy,iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

[0136] The term “substituted alkoxy” refers to the groups—O-(substituted alkyl), —O-(substituted alkenyl), —O-(substitutedcycloalkyl), —O-(substituted cycloalkenyl), —O-(substituted alkynyl) and—O-(optionally substituted alkylene)-alkoxy.

[0137] The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from about 1 to 20 carbonatoms, more preferably about 1 to 10 carbon atoms, and even morepreferably about 1 to 6 carbon atoms. This term is exemplified by groupssuch as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,n-hexyl, n-decyl, tetradecyl, and the like.

[0138] The term “substituted alkyl” refers to:an alkyl group in which 1or more (up to about 5, preferably up to about 3) hydrogen atoms isreplaced by a substituent independently selected from the group: ═O, ═S,acyl, acyloxy, optionally substituted alkoxy, optionally substitutedamino, azido, carboxyl, (optionally substituted alkoxy)carbonyl,(optionally substituted amino)carbonyl, cyano, optionally substitutedcycloalkyl, optionally substituted cycloalkenyl, halogen, hydroxyl,nitro, sulfanyl, sulfinyl, and sulfonyl. One of the preferred optionalsubstituents for alkyl is hydroxy, exemplified by hydroxyalkyl groups,such as 2-hydroxyethyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl,and the like; dihydroxyalkyl groups (glycols), such as2,3-dihydroxypropyl, 3,4-dihydroxybutyl, 2,4-dihydroxybutyl, and thelike; and those compounds known as polyethylene glycols, polypropyleneglycols and polybutylene glycols, and the like. Another preferredsubstitutent for alkyl is optionally substituted alkoxy carbonyl, suchas 1-methoxycarbonyl-ethyl.

[0139] The term “alkylene” refers to a diradical derived from theabove-defined monoradical, alkyl. This term is exemplified by groupssuch as methylene (—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers[e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—] and the like.

[0140] The term “substituted alkylene” refers to a diradical derivedfrom the above-defined monoradical, substituted alkyl. Examples ofsubstituted alkylenes are chloromethylene (—CH(Cl)—), aminoethylene(—CH(NH₂)CH₂—), methylaminoethylene (—CH(NHMe)CH₂—), 2-carboxypropyleneisomers (—CH₂CH(CO₂H)CH₂—), ethoxyethylene (—CH₂CH₂O—CH₂CH₂—),ethyl(N-methyl)aminoethylene (—CH₂CH₂N(CH₃)CH₂CH₂—),1-ethoxy-2-(2-ethoxy-ethoxy)ethylene (—CH₂CH₂O—CH₂CH₂—OCH₂CH₂—OCH₂CH₂—),and the like.

[0141] The term “amino” refers to the group —NH₂.

[0142] The term “substituted amino” refers to the group —NHR or —NRRwhere each R is independently selected from the group: optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted cycloalkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, acyl, optionallysubstituted alkoxy, carboxy and alkoxycarbonyl.

[0143] The term “amino acid” or “natural amino acid” refers to any ofthe twenty (20) common amino acids as generally accepted in the peptideart and represent L-amino acids unless otherwise designated (with theexception of achiral amino acids such as glycine).

[0144] The term “substituted amino acid” refers to an amino acidcontaining one or more additional chemical moieties that are notnormally a part of the amino acid. Such substitutions can be introducedby a targeted deriviatizing agent that is capable of reacting withselected side chains or terminal residues and via other art-acceptedmethods. For example, cysteinyl residues most commonly are reacted with.alpha.-haloacetates (and corresponding amines), such as chloroaceticacid or chloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,

[0145] 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole. Carboxyl side groups (aspartyl orglutamyl) are selectively modified by reaction with carbodiimides(R′—N—C—N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3 (4 azonia 4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions. Glutaminyl andasparaginyl residues are frequently deamidated to the correspondingglutamyl and aspartyl residues. Alternatively, these residues aredeamidated under mildly acidic conditions. Other modifications includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or theonyl residues, methylation of the .alpha.-amino groups oflysine, arginine, and histidine side chains (see, e.g., T. E. Creighton,Proteins: Structure and Molecule Properties, W. H. Freeman & Co., SanFrancisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and,in some instances, amidation of the C-terminal carboxyl groups.

[0146] The term “aromatic” refers to a cyclic or polycyclic moietyhaving a conjugated unsaturated (4n+2)π electron system (where n is apositive integer), sometimes referred to as a delocalized π electronsystem.

[0147] The term “aryl” refers to an aromatic cyclic hydrocarbon group offrom 6 to 20 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferredaryls include phenyl, naphthyl and the like.

[0148] The term “substituted aryl” refers to an aryl group as definedabove, which unless otherwise constrained by the definition for the arylsubstituent, is substituted with from 1 to 5 substituents, andpreferably 1 to 3 substituents, independently selected from the groupconsisting of: ═O, ═S, acyl, acyloxy, optionally substituted alkenyl,optionally substituted alkoxy, optionally substituted alkyl (such astri-halomethyl), optionally substituted alkynyl, optionally substitutedamino, optionally substituted aryl, optionally substituted aryloxy,azido, carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheteroaryl, optionally substituted heteroaryloxy, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, and sulfonyl. Preferred aryl substituents includealkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and sulfinyl.

[0149] The term “aryloxy” refers to the group —O-aryl.

[0150] The term “substituted aryloxy” refers to the group—O-(substituted aryl).

[0151] The term “aralkyl” refers to the moiety “-alkylene-aryl” eachhaving the meaning as defined herein. Such aralkyl groups areexemplified by benzyl, phenethyl, 3-naphthylpropyl and the like.

[0152] The term “substituted aralkyl” refers to the moiety “-(optionallysubstituted aklylene)-(optionally substituted aryl)”, each having themeaning as defined herein, where at least one of the aryl or alkylenegroups is substituted, e.g., 4-(N-methyl-pyrrolyl)pentylene,4-nitrobenzyl or 1-methoxycarbonyl-2-phenyl-ethyl.

[0153] The term “carbonyl” refers to the di-radical “—C(═O)-”, which isalso illustrated as “—C(O)—”.

[0154] The term “(optionally substituted alkoxy)carbonyl” refers to thegroups: —C(O)O-(optionally substituted alkyl), —C(O)O-(optionallysubstituted cycloalkyl), —C(O)O-(optionally substituted alkenyl), and—C(O)O-(optionally substituted alkynyl). These moieties are alsoreferred to as esters.

[0155] The term “(optionally substituted amino)carbonyl” refers to thegroup —C(O)-(optionally substituted amino). This moiety is also referredto as a primary, secondary or tertiary carboxamide.

[0156] The term “(optionally substituted amino)carbonyloxy” refers tothe group —O—C(O)-(optionally substituted amino).

[0157] The term “carboxy” or “carboxyl” refers to the moiety “—C(O)OH”,which is also illustrated as “—COOH”.

[0158] The term “compound of Formula I” is intended to encompass thepyruvate derivatives of the invention as disclosed, and/or thepharmaceutically acceptable salts of such compounds. In addition, thecompounds of this invention include the keto and enol pyruvatetautomers, individual stereochemical isomers (arising from the selectionof substituent groups) and mixtures of tautomers and/or isomers.

[0159] The term “cycloalkyl” refers to non-aromatic cyclic hydrocarbongroups of having about 3 to 40 (preferably about 4 to 15) carbon atomshaving a single ring or multiple condensed rings. Such cycloalkyl groupsinclude, by way of example, single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ringstructures such as adamantanyl, cyclopentaphenanthren and the like.

[0160] The term “substituted cycloalkyl” refers to a cycloalkyl groupsubstituted with from 1 to 5 substituents, and preferably 1 to 3substituents, independently selected from the group consisting of: ═O,═S, acyl, acyloxy, optionally substituted alkenyl, optionallysubstituted alkoxy, optionally substituted alkyl (such astri-halomethyl), optionally substituted alkynyl, optionally substitutedamino, optionally substituted aryl, optionally substituted aryloxy,azido, carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheteroaryl, optionally substituted heteroaryloxy, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, and sulfonyl.

[0161] The term “halo” or “halogen” refers to fluoro, chloro, bromo andiodo.

[0162] The term “heteroaryl” refers to an aromatic cyclic hydrocarbongroup having about 1 to 40 (preferably from about 3 to 15) carbon atomsand about 1 to 10 hetero atoms (preferably about 1 to 4 heteroatoms,selected from nitrogen, sulfur, phosphorus, selenium and/or oxygen)within at least one ring. Such heteroaryl groups can have a single ring(e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinylor benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl,furyl, benzoimidazole, benzothiazole and benzoselenazol.

[0163] The term “substituted heteroaryl” refers to a heteroaryl group asdefined above, which unless otherwise constrained by the definition forthe heteroaryl substituent, is substituted with from 1 to 5substituents, and preferably 1 to 3 substituents, independently selectedfrom the group consisting of: ═O, ═S, acyl, acyloxy, optionallysubstituted alkenyl, optionally substituted alkoxy, optionallysubstituted alkyl (such as tri-halomethyl), optionally substitutedalkynyl, optionally substituted amino, optionally substituted aryl,optionally substituted aryloxy, azido, carboxyl, (optionally substitutedalkoxy)carbonyl, (optionally substituted amino)carbonyl, cyano,optionally substituted cycloalkyl, optionally substituted cycloalkenyl,halogen, optionally substituted heteroaryl, optionally substitutedheteroaryloxy, optionally substituted heterocyclyl, optionallysubstituted heterocyclooxy, hydroxyl, nitro, sulfanyl, sulfinyl, andsulfonyl.

[0164] The term “heteroaralkyl” refers to the moiety“-alkylene-heteroaryl” each having the meaning as defined herein.

[0165] The term “substituted heteroaralkyl” refers to the moiety“-(optionally substituted aklylene)-(optionally substitutedheteroaryl)”, each having the meaning as defined herein.

[0166] The term “heteroaryloxy” refers to the group —O-heteroaryl.

[0167] The term “heteroarylene” refers to the diradical group derivedfrom heteroaryl (including substituted heteroaryl), as defined above,and is exemplified by the groups 2,6-pyridylene, 2,4-pyridiylene,1,2-quinolinylene, 1,8-quinolinylene, 1,4-benzofuranylene,2,5-pyridnylene, 2,5-indolenyl and the like.

[0168] The terms “heterocycle”, “heterocyclic” and “heterocyclyl” referto a monoradical, saturated or unsaturated, non-aromatic cyclichydrocarbon group having about 1 to 40 (preferably from about 3 to 15)carbon atoms and about 1 to 10 hetero atoms (preferably about 1 to 4heteroatoms, selected from nitrogen, sulfur, phosphorus, and/or oxygen)within the ring. Such heterocyclic groups can have a single ring ormultiple condensed rings. Preferred heterocyclics include morpholino,piperidinyl, and the like.

[0169] The terms “substituted heterocycle”, “substituted heterocyclic”and “substituted heterocyclyl” refer to a heterocyclyl group as definedabove, which unless otherwise constrained by the definition for theheterocycle, is substituted with from 1 to 5 substituents, andpreferably 1 to 3 substituents, independently selected from the groupconsisting of: ═O, ═S, acyl, acyloxy, optionally substituted alkenyl,optionally substituted alkoxy, optionally substituted alkyl (such astri-halomethyl), optionally substituted alkynyl, optionally substitutedamino, optionally substituted aryl, optionally substituted aryloxy,azido, carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheteroaryl, optionally substituted heteroaryloxy, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, and sulfonyl.

[0170] The term “heterocycloalkyl” refers to the moiety“-alkylene-heterocycle” each having the meaning as defined herein.

[0171] The term “substituted heterocycloalkyl” refers to the moiety“-(optionally substituted aklylene)-(optionally substitutedheterocycle)”, each having the meaning as defined herein.

[0172] The term “heterocyclooxy” refers to the group —O-heterocycle.

[0173] As used herein, “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0174] The term “pharmaceutically acceptable salt” refers to salts whichretain the biological effectiveness and properties of the compounds ofthis invention and which are not biologically or otherwise undesirable.In many cases, the compounds of this invention are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto. Pharmaceutically acceptablebase addition salts can be prepared from inorganic and organic bases.Salts derived from inorganic bases, include by way of example only,sodium, potassium, lithium, ammonium, calcium and magnesium salts. Saltsderived from organic bases include, but are not limited to, salts ofprimary, secondary and tertiary amines, such as alkyl amines, dialkylamines, trialkyl amines, substituted alkyl amines, di(substituted alkyl)amines, tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines,trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl)amines, tri(substituted alkenyl) amines, cycloalkyl amines,di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkylamines, disubstituted cycloalkyl amine, trisubstituted cycloalkylamines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl)amines, substituted cycloalkenyl amines, disubstituted cycloalkenylamine, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines,triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclicamines, mixed di- and tri-amines where at least two of the substituentson the amine are different and are selected from the group consisting ofalkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,heteroaryl, heterocyclic, and the like. Also included are amines wherethe two or three substituents, together with the amino nitrogen, form aheterocyclic or heteroaryl group.

[0175] Specific examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like.

[0176] Pharmaceutically acceptable acid addition salts may be preparedfrom inorganic and organic acids. Salts derived from inorganic acidsinclude hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

[0177] The term “sulfanyl” refers to the groups: —S-(optionallysubstituted alkyl), —S-(optionally substituted aryl), —S-(optionallysubstituted heteroaryl), —S-(optionally substituted heterocyclyl).Preferred sulfanyl groups include, by way of example, methylsulfanyl(—SCH₃), n-(iso-propylsulfanyl) (—SCH(CH₃)₂) and the like.

[0178] The term “sulfinyl” refers to the groups: —S(O)-(optionallysubstituted alkyl), —S(O)-optionally substituted aryl),—S(O)-(optionally substituted heteroaryl), —S(O)-(optionally substitutedheterocyclyl).

[0179] The term “sulfonyl” refers to the groups: —S(O₂)-(optionallysubstituted alkyl), —S(O₂)-optionally substituted aryl),—S(O₂)-(optionally substituted heteroaryl), —S(O₂)-(optionallysubstituted heterocyclyl).

[0180] The term “therapeutically effective amount” refers to that amountof a compound of Formula I that is sufficient to effect treatment, asdefined below, when administered to a mammal in need of such treatment.The therapeutically effective amount will vary depending upon thesubject and disease condition being treated, the weight and age of thesubject, the severity of the disease condition, the particular compoundof Formula I chosen, the dosing regimen to be followed, timing ofadministration, the manner of administration and the like, all of whichcan readily be determined by one of ordinary skill in the art.

[0181] The term “treatment” or “treating” means any treatment of adisease or disorder in a mammal, including:

[0182] preventing or protecting against the disease or disorder, thatis, causing the clinical symptoms not to develop;

[0183] inhibiting the disease or disorder, that is, arresting orsuppressing the development of clinical symptoms; and/or

[0184] relieving the disease or disorder, that is, causing theregression of clinical symptoms.

[0185] It will be understood by those skilled in the art that in humanmedicine, it is not always possible to distinguish between “preventing”and “suppressing” since the ultimate inductive event or events may beunknown, latent, or the patient is not ascertained until well after theoccurrence of the event or events. Therefore, as used herein the term“prophylaxis” is intended as an element of “treatment” to encompass both“preventing” and “suppressing” as defined herein. The term “protection,”as used herein, is meant to include “prophylaxis.”

[0186] The term “effective amount” means a dosage sufficient to providetreatment for the disorder or disease state being treated. This willvary depending on the patient, the disease and the treatment beingeffected.

[0187] The term “disorder” or “disease state” means any disease,condition, symptom, or indication.

Compounds of the Invention

[0188] The compounds employed in the practice of the present inventionare those identified above with reference to Formulae I, Ia, Ib, Ic, IIand III, and the precursors/intermediates described with reference tothe Reaction Schemes. Formula I is addressed to certain known and novelpyruvate derivatives as employed in novel methods of treatment,pharmaeutical formulations and in the manufacture of medicaments forsuch methods of treatment. Formulae Ia, Ib, Ic, II and III are addressedto novel pyruvate derivatives, methods of treatment, pharmaeuticalformulations and the manufacture of medicaments for such methods oftreatment.

[0189] Nomenclature

[0190] The compounds of the present invention are named and numbered asdescribed below, for example, with reference to Formulae Id, Ie, If, Ig,Ih, and IIIa.

[0191] Formula Id represents the compound according to Formula Ia whereA is 2-thioxo-imidazolidin-1-yl, R is ethyl, X is a covalent bond, wheretautomeric form of the pyruvate (represented by the dashed line inFormula I) is the keto tautomer. In one nomenclature system, thecompound of Formula Id is named:2-oxo-3-(2-thioxo-imidazolidin-1-yl)-propionic acid ethyl ester. Thecompound of Formula Id can also be named as:(2-thioxo-imidazolidin-1-yl)methyl-ketopyruvate ethyl ester.

[0192] Formula Ie represents the compound of Formula Ia where A is3-[2-Amino-9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-9H-purin-6-yl,R is Ethyl, and X is S, which is named:

[0193]3-[2-amino-9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-9H-purin-6-ylsulfanyl]-2-oxo-propionicacid ethyl ester.

[0194] Formula If represents the compound of Formula Ia where A isy-Asp-Cys-Glu, R is ethyl, and X is a Covalent Bond where tautomericform of the pyruvate (represented by the dashed line in Formula I) isthe enol tautomer. This is also the compound of Formula II where R¹ isCOOH, R² is H, R³ is CH₂—S—Pyruvate ethyl ester, R⁴ is optionallysubstituted alkyl where the substituent is COOH, R⁵ is H, k is 0, m is1, and n is 1. The compound of Formula If can be named:2-[2-(3-amino-3-carboxy-propionylamino)-3-(2-ethoxycarbonyl-2-hydroxyvinylsulfanyl)-propionylamino]-pentanedioicacid.

[0195] Formula Ig represents a compound of Formula Ib where A isGlu-Cys-Gly, X is a covalent bond to the sulfur atom of Cys, W is═NOCH₃, and Z is O-ethyl. It is also a compound of Formula II where R¹is COOH, R² is H, R³ is CH₂—S—C(NOCH₃)C(O)OC₂H₆ R⁴ and R⁵ are H, k is 0,m is 2, and n is 1. The compound of Formula Ig can be named2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid.

[0196] Formula Ih represents a compound of Formula Ic where A isGlu-Cys-Gly, X is a covalent bond to the sulfur atom of Cys, W is ═O,and Z is —NR^(b)R^(c) where R^(b) and R^(c) are both ethyl. It is also acompound of Formula II where R¹ is COOH, R² is H, R³ isCH₂—S—C(O)C(O)N(C₂H₆)₂, R⁴ and R⁵ are H, k is 0, m is 2, and n is 1. Thecompound of Formula Ih can be named2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-diethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid.

[0197] Formula IIIa represents a compound of Formula III where R³ ¹ ishydrogen, R^(3.2) is ethyl, R^(3.3) and R^(3.4) are both methyl, and R³⁵ is COOH. The compound of Formula IIIa can be named2,2-dimethyl-3,4-dihydro-2H-[1,4]thiazine-3,5-dicarboxylic acid 5-ethylester.

Synthesis of the Compounds of the Invention

[0198] The compounds of Formulae I and II can be prepared by solutionphase synthesis and, particularly in the case of the compounds ofFormula II, by solid phase supported synthesis. These are described ingreater detail below with reference to the Reaction Schemes.

[0199] Synthetic Reaction Parameters

[0200] The terms “solvent”, “inert organic solvent” or “inert solvent”mean a solvent inert under the conditions of the reaction beingdescribed in conjunction therewith. Solvents employed in synthesis ofthe compounds of the invention include, for example, methanol, acetone,water, acetonitrile, 1,4-dioxane, dimethylformamide (“DMF”), benzene,toluene, tetrahydrofuran (“THF”), chloroform, methylene chloride (ordichloromethane), diethyl ether, pyridine and the like, as well asmixtures thereof. Unless specified to the contrary, the solvents used inthe reactions of the present invention are inert organic solvents.

[0201] The term “q.s.” means adding a quantity sufficient to achieve astated function, e.g., to bring a solution to the desired volume (i.e.,100%).

[0202] Unless specified to the contrary, the reactions described hereintake place at atmospheric pressure within a temperature range from 0° C.to 110° C. (preferably from 0° C. to 25° C.; most preferably at “room”or “ambient” temperature, e.g., 20° C.). Further, unless otherwisespecified, the reaction times and conditions are intended to beapproximate, e.g., taking place at about atmospheric pressure within atemperature range of about 0° C. to about 110° C. (preferably from about0° C. to about 25° C.; most preferably at about “room” or “ambient”temperature, e.g., approximately 20° C.) over a period of about 1 toabout 10 hours (preferably about 5 hours). Parameters given in theExamples are intended to be specific, not approximate.

[0203] Isolation and purification of the compounds and intermediatesdescribed herein can be effected, if desired, by any suitable separationor purification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can, of course, also beused.

BRIEF DESCRIPTION OF REACTION SCHEMES

[0204] Reaction Schemes 1, 1a, 1b and 1c illustrate the solution phasesynthesis of the compounds of Formulae I and II.

[0205] Reaction Schemes 2-6 illustrate the solid phase supportedsynthesis of peptide-pyruvate conjugate compounds of Formulae Ia and II.

[0206] Reaction Scheme 2 illustrates synthesis of peptide precursors tocertain compounds of Formulae I and II, using Fmoc and ^(t)Bocprotecting groups. Solid phase supported synthesis of single aminoacid-pyruvate conjugates, while not preferred, can be accomplished asillustrated and may be found advantageous with certain combinations ofsubstituents.

[0207] Reaction Scheme 3 illustrates the solid phase supported synthesisof peptide-pyruvate conjugates from Fmoc-protected precursors, theirde-protection, cleavage from the solid support, and isolation.

[0208] Reaction Scheme 4 illustrates the solid phase supported synthesisof peptide-pyruvate conjugates from ^(t)Boc-protected precursors, theirde-protection, cleavage from the solid support, and isolation.

[0209] Reaction Schemes 5 and 6 illustrates the solid phase supportedcoupling of different structural moieties onto a precursor's terminalamino group, followed by pyruvate conjugation, de-protection, cleavageand isolation to give peptide-pyruvate conjugates of Formula II where R²is other than hydrogen.

[0210] With regard to Reaction Schemes 3 through 6, it should be noted(for the compounds having more than one amino acid) that the site ofpyruvate conjugation need not be on the illustrated amino acid, but canbe from any of the amino acid positions. Thus, for example, whileFormula 300c illustrates the synthesis of a compound having the pyruvateconjugate at AA₂, the Formula 300c is intended to encompass thecompounds where the —CH₂—S—L group is on AA₁, AA₂ or M₃, i.e., all ofthe following:

[0211] Only a single, representative position for pyruvate conjugationis illustrated for each of the compound in these reaction schemes, theothers having been omitted for the sake of brevity.

[0212] Reaction Scheme 7 illustrates another solid phase supportedapproach for the synthesis and derivitization of resin-imine-pyruvates,as further described in co-pending applications Serial No. 60/288,649,filed May 3, 2001, and Serial No. ______ entitled “PROCESS FOR SOLIDSUPPORTED SYNTHESIS OF PYRUVATE-DERIVED COMPOUNDS” filed on even dateherewith.

[0213] Starting Materials

[0214] The compound ethyl-3-bromopyruvate is commercially available,e.g., from Aldrich Chemical Company, Milwaukee, Wis. N-Fmoc- andN-^(t)Boc-protected amino acids, including S-t-butylthio- andS-trityl-cysteine, are available, e.g., from Advanced ChemTech, Inc. ofLouisville, Ky. Other reactants, such as p-toluenesulfonic acid,3H-imidazole-4-thiol, and solid supports such as Wang resin are likewisecommercially available or may be readily prepared by those skilled inthe art using commonly employed methodology.

[0215] Preparation of Formula I

[0216] Referring to Reaction Scheme 1, approximately equimolarequivalents of a compound of Formula 101 where A and X have the meaningspreviously described, such as:

[0217] an aryl, aralkyl, heteroaryl or heteroaralkyl compound,

[0218] a nucleoside, amino acid, di-, tri- or tetra-peptide,

[0219] an aryl-amide, -thiol, -sulfane, -sulfone,-mercaptopyruvate-thiol,

[0220] an aralkyl-amide, -thiol, -sulfane, -sulfone,-mercaptopyruvate-thiol,

[0221] a heteroaryl-amide, -thiol, -sulfane, -sulfone,-mercaptopyruvate-thiol, or

[0222] a heteroaralkyl-amide, -thiol, -sulfane, -sulfone,-mercaptopyruvate-thiol,

[0223] (any of which compounds of Formula 101 may optionally besubstituted) and a compound of Formula 102 where Z has the meaningpreviously described and L is a leaving group such as a halide(preferably a bromide) together with an appropriate solvent (such asmethanol, acetone, water, acetonitrile, 1,4-dioxane or DMF) arecontacted in a suitable reaction vessel, optionally in the presence ofan organic base (such as a tertiary amine or imidazole). The reactiontakes place at a temperature from 0° C. to 110° C. (preferably 0° C. to25° C.) for 30 minutes to 15 hours (preferably 3-5 hours), followed byremoval of the solvent(s), isolation and purification to give thecorresponding product of Formula I. Additional isolation andpurification steps well known to those skilled in the art may beperformed, e.g., to provide single isomers and/or tautomers.

[0224] Preparation of Formula Ia

[0225] Referring to Reaction Scheme Ia, the compounds of Formula Ia areprepared as described above with reference to Reaction Scheme 1,employing a compound of Formula 102.1 (e.g., an optionally alkyl- oraryl-substituted halopyruvate).

[0226] Preparation of Formula Ib

[0227] Referring to Reaction Scheme Ib, a compound of Formula 102.1 iscontacted with a compound of the formula R^(a)ONH₂ and converted to thecorresponding oxime of Formula 102.2, which is then contacted with acompound of Formula 101 as described above with reference to ReactionScheme 1. Alternatively, the compound of the formula R^(a)ONH₂ can beemployed with a compound of Formula Ia to give the corresponding oximeof Formula Ib. Reductive alkylation or acylation of a compound ofFormula Ib can be employed to obtain the corresponding compounds ofFormula I where W is —N(OH)—R^(d).

[0228] In like fashion, reaction of a compound of Formula Ia with acompound of the formula R^(b)R^(c)NNH₂ will give the correspondingcompounds of Formula I where W is ═N—NR^(b)R^(c).

[0229] Preparation of Formula Ic

[0230] Referring to Reaction Scheme 1 c, a pyruvic acid of Formula 102.3is contacted with a secondary amine of the formula HNR^(b)R^(c) to givethe corresponding compound of Formula 102.4, which can be converted tothe corresponding oxime of Formula 102.5 via reaction with a compound ofFormula R^(a)ONH₂. The compounds of Formulae 102.4 and 102.5 can beconverted to the corresponding compounds of Formula Ic by raction with acompound of Formula 101 (A-XH) as described above.

[0231] Similarly, starting with a compound of Formula Ic where W is O,reaction with R^(a)ONH₂ or R^(b)R^(c)NNH₂ will give the correspondingcompounds of Formula Ic where W is ═N—O—R^(a) or ═N—NR^(b)R^(c).Reductive alkylation or acylation of a compound of Formula I c can beemployed to obtain the corresponding compounds of Formula I where W is—N(OH)—R^(d).

[0232] Preparation of Formula II

[0233] The compounds of Formula II, particularly polypeptides can beprepared, with few exceptions, using solid phase support synthesismethods. These are illustrated with reference to Reaction Schemes 2through 6.

[0234] As illustrated in Reaction Scheme 2, Step 2.1, a solid support201 (such as Wang resin) and a symmetrical Fmoc-protected amino acidanhydride 202 are linked employing Fmoc coupling/de-protecting protocolsknown in the art, to give the corresponding resin-bound, Fmoc-protectedamino acid of Formula 203. For example, to approximately 10 molarequivalents of GlyOH dissolved in DCM is added DIC (5 eq) in smallportion with stirring. Stirring is continued for 1 hour, after which thesolution is added to the Wang resin (1 eq, pre-swelled in DMF) in thepresence of 0.1 molar equivalent of DMAP. The resin suspension is shakenfor 1 hour, followed by a thorough wash with DMF. The resulting preparedresin 201 is coupled with 2 molar equivalents of amino acid anhydride202 using TBTU (2 eq), DIPEA (4 eq), followed by DMF wash (3 times).Formula 203 is de-protected (as shown in Step 2.2), e.g., using 20%piperidine in DMF followed by a DMF wash (5 times), to give theresin-bound amino acid of Formula 204, which may be linked to additionalamino acids (as shown in Step 2.3) or conjugated to pyruvate (as shownin Reaction Scheme 3).

[0235] A resin-bound amino acid of Formula 204 is coupled with aprotected amino acid, such as 205, to give resin-bound, Fmoc-protecteddi-peptide of Formula 206, which is de-protected to give thecorresponding resin-bound di-peptide of Formula 207, which may be linkedto additional amino acids (as shown in Step 2.5) or conjugated topyruvate (as shown in Reaction Scheme 3). The reactions take place underconditions similar to those discussed above with respect to Steps 2.1and 2.2. (Formula 205 is illustrated as Fmoc protected, but as will beapparent to those skilled in the art, may optionally beN-^(t)Boc-protected).

[0236] As illustrated in Step 2.5, the N-terminal amino acid unit (e.g.,AA₃) can be either N-Fmoc- or N-^(t)Boc-protected. A resin-bounddi-peptide of Formula 207 is coupled with a protected amino acid, suchas 208, to give resin-bound, protected tri-peptide of Formula 209. Thereaction takes place under conditions similar to those discussed abovewith respect to Step 2.3. As illustrated in Step 2.6, N-Fmoc-protectedtripeptides of Formula 209 are de-protected under conditions similar tothose discussed above with respect to Step 2.4, and then conjugated topyruvate (e.g., as shown in Reaction Scheme 3). N-^(t)Boc-protected,resin-linked peptides (such as those of Formulae 206 or 209) are carriedforward as illustrated in Reaction Scheme 4.

[0237] Reaction Scheme 3 describes a synthetic route to thepeptide-pyruvate conjugates starting from previously N-Fmoc-protectedresin-linked peptide precursors, such as those of Formulae 204, 207 and210 (corresponding to starting compounds 300a, 300b and 300c), which arerespectively illustrated as having a protected Cysteine at AA₁, AA₂ andAA₂, respectively (the protecting group, e.g., t-Butyl or trityl beingdesignated as “L”). The protecting groups are introduced with thecorresponding amino acid in Steps 2.1, 2.3 and/or 2.5. It will beappreciated that any of amino acids AA₁, AA₂ and AA₃ (or a fourth aminoacid, not shown) can be Cysteine as employed in Reaction Schemes 3, 4, 5and 6, to give the corresponding peptide-pyruvate conjugates at AA₁, AA₂and/or AA₃.

[0238] The cysteine of a resin-linked peptide, such as 300a, 300b and300c, as illustrated in Step 3.1, is de-protected by treating withdithiothreitol to give the corresponding compound of Formula 301 a, 301b or 301 c. This is followed by conjugation of the de-protected thiogroup with two molar equivalents of a halopyruvate of Formula 102 bynucleophilic substitution, as illustrated in Step 3.2. The resultingresin-bound peptide-pyruvate conjugate of Formula 302a, 302b or 302c isthen cleaved from the resin under acidic conditions, e.g., with 95% TFA(aq.), isolated and then purified by typical methods (e.g., cold etherwash, filtration and lyophilization) to give the corresponding freepeptide-pyruvate conjugate of Formula 303a, 303b or 303c.

[0239] Reaction Scheme 4 describes a synthetic route to thepeptide-pyruvate conjugates starting from N-^(t)Boc-protectedresin-linked peptide precursors, such as those of Formulae 206 and 209(corresponding to starting compounds 400b and 400c). Synthesis startingfrom an N-^(t)Boc-protected amino acid is illustrated with reference tocompound 400a.

[0240] As in Reaction Scheme 2, the cysteine of a resin-linked peptide,such as 400a, 400b and 400c, as illustrated in Step 4.1, is de-protectedby treating with dithiothreitol to give the corresponding compound ofFormula 401a, 401 b or 401 c, followed by conjugation of thede-protected thio group with two molar equivalents of a halopyruvate ofFormula 102 (Step 4.2). The resulting N-^(t)Boc-protected resin-boundpeptide-pyruvate conjugate of Formula 402a, 402b or 402c is thendeprotected and cleaved from the resin under acidic conditions, e.g.,with 95% TFA (aq.), isolated and then purified by typical methods (asabove) to give the corresponding free peptide-pyruvate conjugate ofFormula 403a, 403b or 403c.

[0241] Reaction Scheme 5 illustrates the coupling of various structuralmoieties onto the amino group of the peptide-pyruvate conjugates of theinvention, utilizing solid phase support synthesis. These compounds arefurther described with reference to Examples 23-27.

[0242] As illustrated in Step 5.1, starting from previouslyN-Fmoc-protected resin-linked peptide precursors, such as those ofFormulae 204, 207 and 210 (corresponding to starting compounds 500a,500b and 500c), coupling of a diphenol acid 501 to the amino group ofthe pyruvate-peptide is effected by using pre-activated HOBt ester(using DIC as a dehydrating agent) to give the corresponding compoundsof Formulae 502a, 502b, and 502c. The thio protecting group is thenremoved (Step 5.2), followed by pyruvate conjugation (Step 5.3),cleavage, isolation and purification (Step 5.4) as described above withregard to Reaction Scheme 3 to give the corresponding compounds of theinvention identified as Formulae 505a, 505b and 505c.

[0243] Reaction Scheme 6 illustrates an alternative sequence forcoupling structural moieties onto the amino group of thepeptide-pyruvate conjugates of the invention, beginning with cleavage ofa precursor (here a diphenol acid coupled compound of Formula 503a, 503bor 503c) from the resin (Step 6.1), followed by pyruvate coupling,isolation and purification (Step 6.2). These reactions are carried outunder conditions similar to the respective steps in Reaction Scheme 5.

[0244] An alternative solid supported approach for the synthesis ofcompounds of Formula I is illustrated with respect to Reaction Scheme 7.

Preferred Processes and Last Steps

[0245] The preferred process for generating compounds of Formula Ia, Ib,Ic and III is solution phase reaction, e.g., as exemplified for thesynthesis of Example 1-16. This process represents a simple and directsynthetic route in which the A-X in Scheme 1 is readily available. Apreferred process for the preparation of compounds, particularly ofFormula II, is the solid phase supported synthesis approach, e.g., asexemplified in the synthesis of Examples 16, and 19 to 27. It allows forthe synthesis of structure-defined and more complex conjugates. Thisparticularly allows for preparation of a large number of structurallydiverse molecules using a parallel approach, but would not be preferredfor the synthesis of large amounts of a particular compound.

[0246] Thus, in one preferred aspect, a bromopyruvate and athio-containing nucleophile are contacted and subjected to conditionsfor nucleophilic substitution.

[0247] In another preferred aspect, a solid phase supported pyruvateconjugate is cleaved from the support.

[0248] In still another preferred aspect, a solid support-freecystein-containing peptide is conjugated with a bromopyruvate.

[0249] A compound of Formula I is contacted with a pharmaceuticallyacceptable acid to form the corresponding acid addition salt.

[0250] A pharmaceutically acceptable acid addition salt of Formula I iscontacted with a base to form the corresponding free base of Formula I.

Preferred Compounds

[0251] In a preferred embodiment where A is a natural or substitutedamino acid or peptide, A is selected from the group: Ala, Asn, Asp, Cys,Gin, Glu, Gly, Lys, Met, Ser and Thr, especially Ala, Asp, Cys, Glu andGly. Further preferred are those compounds where A is a natural orsubstituted di- or tri-peptide, especially natural peptides. Mostpreferred is the tri-peptide Glu-Cys-Gly.

[0252] In another preferred embodiment, A is an optionally substitutedheteroaryl group, especially a nitrogen-containing optionallysubstituted heteroaryl, and particularly where A is selected from thegroup: imidazole, triazole, thiadiazole, oxadiazole, benzoselenazole,benzoimidazole and benzothiazole.

[0253] Further preferred in each of the foregoing embodiments are thosecompounds where X is —S— or a covalent bond.

[0254] With overall regard to Formulae I, Ia, Ib and Ic, preferred arethose compounds, pharmaceutical formulations, methods of treatment andthe manufacture of medicaments having the following combinations andpermutations of substituent groups (sub-grouped, respectively, inincreasing order of preference, each sub-grouping being intended ascombinable with other sub-groupings):

[0255] A is optionally substituted alkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted heteroaryl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkyl, an optionally substituted amino acid, or an optionallysubstituted di-, tri- or tetra-peptide.

[0256] Particularly where A is: an optionally substituted amino acidselected from Ala, Asp, Cys, Glu and Gly, or an optionally substituteddi- or tri-peptide the amino acids of which are selected from Ala, Asp,Cys, Glu and Gly.

[0257] Preferably where A is the tri-peptide Glu-Cys-Gly.

[0258] More preferably where X is a covalent bond to the sulfur atom ofCys.

[0259] Preferably where A is substituted alkyl selected from:—CH₂—CH(OH)—CH₂—OH, and —CH(CH₃)—C(O)—N(H)—CH₂—COOH); optionallysubstituted heteroaryl selected from: benzoselenazol-2-yl, 5-(chloro ormethoxy)-substituted-1H-benzoimidazol-2-yl, and 5-(chloro, methoxy ornitro)-substituted-benzothiazol-2-yl; heterocyclyl selected from:4,5-dihydro-thiazol-2-yl, 2-thioxo-imidazolidin-1-yl and morpholino; oran optionally substituted di-, tri- or tetra-peptide.

[0260] Particularly where X is —S— or or a covalent bond to the sulfuratom of Cys or to the nitrogen atom of optionally substitutedheterocyclyl.

[0261] Particularly where W is ═O or ═N—OR^(a).

[0262] Particularly where Z is —OR or —NR^(b)R^(c).

[0263] W is ═O or ═N—OR^(a).

[0264] Particularly where Z is —OR or —NR^(b)R^(c).

[0265] Particularly where W is ═O and Z is —OR

[0266] Preferably where R is hydrogen or C₁ to C₈ alkyl.

[0267] More preferably where R is hydrogen, ethyl or n-butyl.

[0268] Preferably where A is substituted alkyl selected from:

—CH₂—CH(OH)—CH₂—OH, and —CH(CH₃)—C(O)—N(H)—CH₂—COOH);

[0269]  optionally substituted heteroaryl selected from:benzoselenazol-2-yl, 5-(chloro ormethoxy)-substituted-1H-benzoimidazol-2-yl, and 5-(chloro, methoxy ornitro)-substituted-benzothiazol-2-yl; heterocyclyl selected from:4,5-dihydro-thiazol-2-yl, 2-thioxo-imidazolidin-1-yl and morpholino; oran optionally substituted di-, tri- or tetra-peptide.

[0270] More preferably where X is —S— or or a covalent bond to thesulfur atom of Cys or to the nitrogen atom of optionally substitutedheterocyclyl.

[0271]  Most preferably where R is hydrogen or C₁ to C₈ alkyl.

[0272] Preferably where X is —S— or or a covalent bond to the sulfuratom of Cys or to the nitrogen atom of optionally substitutedheterocyclyl.

[0273] Particularly where W is ═O and Z is —NR^(b)R^(c).

[0274] Preferably where R^(b) and R^(c) are C₁ to C₄ alkyl.

[0275] Preferably where R^(b) is C₁ to C₈ optionally acyl-substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl orcycloalkyl, and R^(c) is hydrogen.

[0276] Preferably where R^(b) and R^(c) together with the nitrogen towhich they are attached form a 2-optionally substituted-pyrrolidine ringor a 6-membered ring selected from 4-optionallysubstituted-piperidin-1-yl and morpholin-4-yl.

[0277] Particularly where W is ═N—OR^(a).

[0278] Preferably where R^(a) is hydrogen, C₁ to C₄ alkyl or alkenyl,phenyl or optionally substituted benzyl.

[0279] Preferably where Z is —OR or —NR^(b)R^(c).

[0280] More preferably where R is hydrogen or C₁ to C₈ alkyl.

[0281]  Most preferably where R is hydrogen, ethyl or n-butyl.Preferably where Z is —NR^(b)R^(c).

[0282] More preferably where R^(b) and R^(c) are C₁ to C₄ alkyl.

[0283] More preferably where R^(b) is C₁ to C₈ optionallyacyl-substituted alkyl, optionally substituted aryl, optionallysubstituted aralkyl or cycloalkyl, and R^(c) is hydrogen.

[0284] More preferably where R^(b) and R^(c) together with the nitrogento which they are attached form a 2-optionally substituted-pyrrolidinering or a 6-membered ring selected from 4-optionallysubstituted-piperidin-1-yl and morpholin-4-yl.

[0285] Preferably where A is substituted alkyl selected from:

—CH₂—CH(OH)—CH₂—OH, and —CH(CH₃)—C(O)—N(H)—CH₂—COOH);

[0286]  optionally substituted heteroaryl selected from: 5-(chloro ormethoxy)-substituted- 1H-benzoimidazol-2-yl, and 5-(chloro, methoxy ornitro)-substituted-benzothiazol-2-yl; heterocyclyl selected from:4,5-dihydrothiazol-2-yl, 2-thioxo-imidazolidin-1-yl and morpholino; oran optionally substituted di-, tri- or tetra-peptide.

[0287] More preferably where X is —S— or or a covalent bond to thesulfur atom of Cys or to the nitrogen atom of optionally substitutedheterocyclyl.

[0288] Z is —OR or —NR^(b)R^(c).

[0289] Especially where R is hydrogen or C₁ to C₈ alkyl.

[0290] Particularly where R is hydrogen, ethyl or n-butyl.

[0291] Especially where R^(b) and R^(c) are C₁ to C₄ alkyl.

[0292] Especially where R^(b) is C₁ to C₈ optionally acyl-substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl orcycloalkyl, and R^(c) is hydrogen.

[0293] Especially where R^(b) and R^(c) together with the nitrogen towhich they are attached form a 2-optionally substituted-pyrrolidine ringor a 6-membered ring selected from 4-optionallysubstituted-piperidin-1-yl and morpholin-4-yl.

[0294] Especially where W is ═N—OR^(a).

[0295] Especially where Z is —NR^(b)R^(c).

[0296] Particularly where W is ═N—OR^(a).

[0297] Preferably whereR^(b) is: C₁ to C₄ alkyl, optionally substitutedaryl, optionally substituted aralkyl or cycloalkyl; and

[0298] Preferably where R^(c) is: hydrogen or C₁ to C₄ alkyl; or

[0299] Preferably where R^(b) and R^(c) together with the nitrogen towhich they are attached form an optionally substituted-pyrrolidine ringa 6-membered ring, optionally incorporating O or N as an additional ringheteroatom, and said ring being optionally substituted with onesubstituent selected from the group consisting of acyl and optionallysubstituted alkyl.

[0300] With regard to Formula II, preferred are those compounds,pharmaceutical formulations, methods of treatment and the manufacture ofmedicaments having the following combinations and permutations ofsubstituent groups (sub-grouped, respectively, in increasing order ofpreference, each sub-grouping being intended as combinable with othersub-groupings):

[0301] R¹ is: —C(O)—O—R′

[0302] Especially where R′ is hydrogen or lower alkyl.

[0303] Especially where R² is hydrogen.

[0304] Especially where R³ is —CH₂—S—CH₂—C(W)—C(O)—Z,—CH₂—S—CH═C(OH)—C(O)—Z, —CH₂—S(O)—CH₂—C(W)—C(O)—Z, or—CH₂—S(O)—CH═C(OH)—C(O)—Z.

[0305] Particularly where W is ═O or ═N—OR^(a).

[0306] Preferably where R^(a) is hydrogen or alkyl;

[0307] Particularly where Z is —OR or —NR^(b)R^(c).

[0308] Preferably where R is hydrogen, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted aryl, oroptionally substituted aralkyl.

[0309] Preferably where R^(b) is C₁ to C₄ alkyl, phenyl or benzyl;.

[0310] Preferably where R^(c) is hydrogen or C₁ to C₄ alkyl.

[0311] Preferably where R^(b) and R^(c) together with the nitrogen towhich they are attachwed form a 6-membered ring selected from4-optionally substituted-piperidin-1-yl and morpholin-4-yl.

[0312] Especially where R⁴ is hydrogen.

[0313] Especially where R⁵ is hydrogen or lower alkyl.

[0314] Especially where k, m and n are respectively: 0,2,1; 1,0,1; or2,0,1.

[0315] With regard to Formula III, preferred are those compounds,pharmaceutical formulations, methods of treatment and the manufacture ofmedicaments having the following combinations and permutations ofsubstituent groups (sub-grouped, respectively, in increasing order ofpreference, each sub-grouping being intended as combinable with othersub-groupings):

[0316] R³¹ is hydrogen

[0317] Especially where R^(3.2) is hydrogen or ethyl.

[0318] Particularly where R^(3.3) and R^(3.4) are both H or are bothmethyl.

[0319] Preferably where R³ ⁵ is: COOH.

[0320] Especially where R³ ³ and R^(3.4) are both H or are both methyl.

[0321] Especially where R^(3.5) is: COOH.

[0322] R^(3.2) is hydrogen or ethyl.

[0323] Especially where R³ ³ and R^(3.4) are both H or are both methyl.

[0324] Especially where R³ ⁵ is: COOH.

[0325] R^(3.3) and R^(3.4) are both H or are both methyl

[0326] Especially where R³ ⁵ is: COOH.

[0327] R^(3.5) is: COOH.

[0328] In the methods of treatment and the manufacture of medicamentsemploying compounds according to Formula I, preferred are thosecompounds the substituents of which are selected from the followinggroups:

[0329] A is: optionally substituted alkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted heteroaryl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkyl, an optionally substituted amino acid, or an optionallysubstituted di-, tri- or tetra-peptide;

[0330] X is: —N(H)—, —S—, or a covalent bond to the sulfur atom of Cysor to the nitrogen atom of optionally substituted heterocyclyl;

[0331] W is: ═O or ═N—OR^(a);

[0332] Z is: —OR, or —NR^(b)R^(c);

[0333] R is: hydrogen, optionally substituted alkyl, substitutedcycloalkyl, or optionally substituted aralkyl;

[0334] R^(a) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, or optionally substituted aralkyl;

[0335] R^(b) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl or optionallysubstituted cycloalkyl;

[0336] R^(c) is: hydrogen or optionally substituted alkyl; and

[0337] R^(b) and R^(c) together with the nitrogen to which they areattached may form a 5- or 6-membered ring, optionally incorporating N orO as an additional ring heteroatom, and said ring being optionallysubstituted with one substituent selected from the group consisting ofacyl and optionally substituted alkyl.

[0338] More preferably, the above substituents of Formula I are furtherselected from the following groups:

[0339] A is: the tri-peptide Glu-Cys-Gly; and

[0340] X is: a covalent bond to the sulfur atom of Cys.

[0341] R is: hydrogen or C₁ to C₈ alkyl;

[0342] R^(a) is: hydrogen, C₁ to C₈ alkyl or alkenyl, phenyl or aralkyl;

[0343] R^(b) is: C₁ to C₈ optionally acyl-substituted alkyl, optionallysubstituted aralkyl or cycloalkyl; and

[0344] R^(c) is: hydrogen or C₁ to C₄ alkyl; or R^(b) and R^(c) togetherwith the nitrogen to which they are attached form a 5-membered ring, ora 6-membered ring optionally incorporating 0 as an additional ringheteroatom, and said ring being optionally substituted with onesubstituent selected from the group consisting of acyl and optionallysubstituted alkyl.

[0345] With regard to Formula Ia, preferred are those compounds thesubstituents of which are selected from the following groups:

[0346] A is: substituted alkyl selected from: —CH₂—CH(OH)—CH₂—OH,—CH(CH₃)—CH(OH)—CH₂—OH, —CH(CH₃)—C(O)—N(H)—CH₂—COOH,—CH₂—C(O)—N(H)—CH₂—COOH, —CH₂—CH₂—C(O)—N(H)—CH₂—COOH,—CH(CH₃)—CH₂—C(O)—N(H)—CH₂—COOH, and —CH₂—CH(CH₃)—C(O)—N(H)—CH₂—COOH,

[0347] substituted heteroaryl selected from:5-chloro-1H-benzoimidazol-2-yl, 5-methoxy-1H-benzoimidazol-2-yl,4-oxo-3,4-dihydro-quinazolin-2-yl, benzoselenazol-2-yl, and5-substituted-benzothiazol-2-yl,

[0348] heterocyclyl selected from: thiazol, 2-thioxo-imidazolidin-1-yland morpholino, or an optionally substituted di-, tri- or tetra-peptide;

[0349] R is: hydrogen, optionally substituted alkyl, or optionallysubstituted cycloalkyl;

[0350] X is: —S—, —S(O)—, —S(O)₂—, or a covalent bond to the sulfur atomof Cys or to the nitrogen atom of optionally substituted heterocyclyl;and

[0351] Z is: —OR.

[0352] More preferably, the above substituents of Formula Ia are furtherselected from the following groups:

[0353] A is: an optionally substituted di- or tri-peptide the aminoacids of which are selected from Ala, Asp, Cys, Glu and Gly; mostpreferably the tri-peptide Glu-Cys-Gly;

[0354] X is: —S—, or a covalent bond to the sulfur atom of Cys or to thenitrogen atom of optionally substituted heterocyclyl; and

[0355] R is: hydrogen or C₁ to C₈ alkyl.

[0356] With regard to Formula Ib, preferred are those compounds thesubstituents of which are selected from the following groups:

[0357] A is: optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, an optionallysubstituted amino acid, or an optionally substituted di-, tri- ortetra-peptide;

[0358] R is: hydrogen, optionally substituted alkyl, substitutedcycloalkyl, or optionally substituted aralkyl;

[0359] R^(a) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, or optionally substituted aralkyl;

[0360] X is: —S—, or a covalent bond to the sulfur atom of Cys or to thenitrogen atom of optionally substituted heterocyclyl; and

[0361] Z is: —OR. More preferably, the above substituents of Formula Ibare further selected from the following groups:

[0362] A is: phenyl or optionally substituted heteroaryl selected from:5-optionally substituted-benzothiazol-2-yl and 5-optionally substitutedbenzoimidazol-2-yl, or an optionally substituted di- or tri-peptide theamino acids of which are selected from Ala, Asp, Cys, Glu and Gly; mostpreferably the tri-peptide Glu-Cys-Gly;

[0363] R^(a) is: hydrogen, C₁ to C₄ alkyl or alkenyl, phenyl oroptionally substituted benzyl; and

[0364] R is: hydrogen or C₁ to C₆ alkyl.

[0365] With regard to Formula Ic, preferred are those compounds thesubstituents of which are selected from the following groups:

[0366] A is: optionally substituted alkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted heteroaryl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkyl, an optionally substituted amino acid, or an optionallysubstituted di-, tri- or tetra-peptide;

[0367] X is: —S—, or a covalent bond to the sulfur atom of Cys;

[0368] W is: ═O or ═N—OR^(a);

[0369] R^(a) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, or optionally substituted aralkyl;

[0370] R^(b) is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl or optionallysubstituted cycloalkyl;

[0371] R^(c) is: hydrogen or optionally substituted alkyl; and

[0372] R^(b) and R^(c) together with the nitrogen to which they areattached may form a 5- or 6-membered ring, optionally incorporating N orO as an additional ring heteroatom, and said ring being optionallysubstituted with one substituent selected from the group consisting ofacyl and optionally substituted alkyl.

[0373] More preferably, the above substituents of Formula Ic are furtherselected from the following groups:

[0374] A is: optionally substituted aryl selected from: phenyl andp-tolyl; or optionally substituted heteroaryl selected from:5-optionally substituted-benzothiazol-2-yl and 5-optionally substitutedbenzoimidazol-2-yl, or an optionally substituted di- or tri-peptide theamino acids of which are selected from Ala, Asp, Cys, Glu and Gly; mostpreferably the tri-peptide Glu-Cys-Gly;

[0375] W is ═O, ═N—OH, or ═N—O—CH₃; and

[0376] R^(b) and R^(c) are C₁ to C₄ alkyl;

[0377] R^(b) is C₁ to C₈ optionally acyl-substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl or cycloalkyl, andR^(c) is hydrogen; or

[0378] R^(b) and R^(c) together with the nitrogen to which they areattached form a 2-optionally substituted-pyrrolidine ring or a6-membered ring selected from 4-optionally substituted-piperidin-1-yland morpholin-4-yl.

[0379] With regard to Formula II, preferred are those compounds thesubstituents of which are selected from the following groups:

[0380] R¹ is: —C(O)—O—R′ where R′ is hydrogen or lower alkyl;

[0381] R² is: hydrogen;

[0382] R³ is: —CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z

[0383] R⁴ is: hydrogen;

[0384] R⁵ is hydrogen or lower alkyl;

[0385] W is: ═O or ═N—OR^(a);

[0386] Z is: —OR or —NR^(b)R^(c);

[0387] R is: hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, or optionallysubstituted aralkyl;

[0388] R^(a) is: hydrogen or alkyl;

[0389] R^(b) is: C₁ to C₄ alkyl, phenyl or benzyl;

[0390] R^(c) is: hydrogen or C₁ to C₄ alkyl, or

[0391] R^(b) and R^(c) together with the nitrogen to which they areattachwed form a 6-membered ring selected from 4-optionallysubstituted-piperidin-1-yl and morpholin-4-yl; and

[0392] k, m and n are respectively: 0,2,1; 1,0,1; or 2,0,1.

[0393] More preferably, the above substituents of Formula II are furtherselected from the following groups:

[0394] R¹ is: —COOH;

[0395] R⁵ is: hydrogen; and

[0396] k, m and n are respectively: 0,2,1; or 2,0,1.

[0397] With regard to Formula III, preferred are those compounds thesubstituents of which are selected from the following groups:

[0398] R^(3.1) is hydrogen;

[0399] R^(3.2) is hydrogen or ethyl;

[0400] R^(3.3) and R^(3.4) are both H or are both methyl; and

[0401] R^(3.5) is: COOH.

[0402] More preferably, the above substituents of Formula III arefurther selected where R^(3.2) is ethyl.

[0403] One series of preferred compounds includes the following, as wellas their stereoisomers, tautomers, salts, and mixtures thereof:

[0404] 3-(1H-Benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester

[0405] 3-(5-Methyl-1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0406] 3-(5-Methoxy-1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0407] 3,4-Dihydro-2H-[1,4]thiazine-3,5-dicarboxylic acid 5-ethyl ester

[0408]1-(2-Carboxy-2-oxo-ethyl)-4-[2-(3,4-dihydroxy-phenyl)-vinyl]-pyridinium;bromide

[0409] 3-(4,5-Dihydro-1H-imidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0410] 2-Hydroxy-3-(1H-imidazol-2-ylsulfanyl)-5-oxo-hex-2-enedioic aciddiethyl ester

[0411]3-[2-Amino-9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-9H-purin-6-ylsulfanyl-2-oxo-propionicacid ethyl ester

[0412] 2-Oxo-3-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-propionic acidethyl ester

[0413] 3-(5-Amino-2H-[1,2,4]triazol-3-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0414] 3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0415] 3-(5-Nitro-1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0416] 2-Oxo-3-(5-phenyl-[1,3,4]oxadiazol-2-ylsulfanyl)-propionic acidethyl ester

[0417] 3,4-Dihydro-2H-[1,4]thiazine-3,5-dicarboxylic acid

[0418]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0419]2-Amino-N-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethyl]-succinamicacid

[0420]3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacid

[0421]3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacid ethyl ester

[0422]2-Amino-4-[2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-1-(methoxycarbonylmethylcarbamoyl)-ethylcarbamoyl]-butyricacid methyl ester

[0423]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-decyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0424]3-[2-(3-Amino-3-carboxy-propionylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacid ethyl ester

[0425]3-{2-Amino-2-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyvinylsulfanyl)-ethylcarbamoyl]-ethylsulfanyl}-2-hydroxy-acrylicacid ethyl ester

[0426]3-[2-(2-Amino-3-mercapto-propionylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylic acid ethyl ester

[0427]4-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)

[0428] -ethylcarbamoyl]-butyric acid

[0429]1-(Carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-{4-[2-(3,4-dihydroxy-phenyl)-vinyl]-benzoylamino}-butyricacid

[0430]4-[1-(Carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-2-{4-[2-(3,4-dihydroxyphenyl)-vinyl]-benzoylamino}-butyricacid

[0431]4-[1-(Carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-[3-(6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionylamino]-butyricacid

[0432]4-[1-(Carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-2-[3-(6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionylamino]-butyricacid

[0433] 3-(5-Methyl-[1,3,4]thiadiazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0434] 3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester

[0435] 3-(4,5-Dihydro-thiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester

[0436]2-Hydroxy-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-2,3-dihydro-furan-2,5-dicarboxylicacid diethyl ester

[0437] 2,2-Dimethyl-3,4-dihydro-2H-[1,4]thiazine-3,5-dicarboxylic acid

[0438]4-[2-[2-(Adamantan-1-ylmethoxycarbonyl)-2-oxo-ethylsulfanyl]-1-(carboxymethylcarbamoyl)-ethylcarbamoyl]-2-amino-butyricacid

[0439]1-[3-(2-Ethoxycarbonyl-2-oxo-ethylsulfanyl)-2-methyl-propionyl]-pyrrolidine-2-carboxylicacid

[0440]2-Amino-3-[1-(2-ethoxycarbonyl-2-oxo-ethyl)-1H-imidazol-4-yl]-propionicacid

[0441]3-[5-(2-Ethoxycarbonyl-2-oxo-ethylsulfanyl)-[1,3,4]thiadiazol-2-ylsulfanyl]-2-oxo-propionicacid ethyl ester

[0442] 2-Oxo-3-(3-phenyl-[1,2,4]oxadiazol-5-ylsulfanyl)-propionic acidethyl ester

[0443] 3-(6-Ethoxy-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester

[0444] 2-Oxo-3-(9H-purin-6-ylsulfanyl)-propionic acid ethyl ester

[0445]3-[9-(3,4-Dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-9H-purin-6-ylsulfanyl]-2-oxo-propionicacid ethyl ester

[0446] 2-Acetylamino-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionicacid

[0447]3-Amino-N-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethyl]-succinamicacid

[0448]2-[2-Amino-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionylamino]-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionicacid

[0449]2-[2-(4-Amino-4-carboxy-butyrylamino)-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionylamino]-4-methylsulfanyl-butyricacid

[0450]2-Amino-4-[1-[1-carboxy-2-(1H-imidazol-4-yl)-ethylcarbamoyl]-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0451]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-octadecyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0452]2-Amino-4-[1-[1-carboxy-2-(1H-indol-2-yl)-ethylcarbamoyl]-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0453]2-Amino-4-[1-[1-carboxy-2-(4-hydroxy-phenyl)-ethylcarbamoyl]-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0454]2-[2-(4-Amino-4-carboxy-butyrylamino)-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionylamino]-3-methyl-butyricacid

[0455]2-Amino-4-[1-(1-carboxy-ethylcarbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0456]2-[2-(4-Amino-4-carboxy-butyrylamino)-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionylamino]-pentanedioicacid

[0457]2-Amino-4-[1-(1-carboxy-2-hydroxy-ethylcarbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0458]2-Amino-4-[1-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0459]2-Amino-4-[1-[1-carboxy-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0460]2-Amino-4-[1-carboxy-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0461]2-Amino-4-[1-(ethoxycarbonylmethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0462]3-[2-Amino-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionicacid ethyl ester

[0463] 2-oxo-3-(2-thioxo-imidazolidin-1-yl)-propionic acid ethyl ester

[0464]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid

[0465]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(3-morpholin-4-yl-2,3-dioxo-propylsulfanyl)-ethylcarbamoyl]-butyricacid

[0466]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0467]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-pyrrolidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid

[0468]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-octylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0469]1-{3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionyl}-pyrrolidine-2-carboxylicacid methyl ester

[0470]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-cyclohexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0471]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(1-methoxycarbonyl-2-phenylethylcarbamoyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid

[0472]2-Amino-4-[2-(2-benzylcarbamoyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid

[0473]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0474]2-{3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionylamino}-3-methyl-pentanoicacid methyl ester

[0475]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-dimethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0476]2-Amino-4-(1-(carboxymethyl-carbamoyl)-2-{2-[2-(4-hydroxy-phenyl)-1-methoxycarbonyl-ethylcarbamoyl]-2-oxo-ethylsulfanyi}-ethylcarbamoyl)-butyricacid

[0477]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-diethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0478]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(4-methyl-cyclohexylcarbamoyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid

[0479]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0480]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0481]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-ethoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0482]2-Amino-4-[2-(2-tert-butoxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid

[0483]4-[2-(2-Allyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-2-amino-butyricacid

[0484]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-ethoxycarbonyl-2-(4-nitro-benzyloxyimino)-ethylsulfanyl]-ethylcarbamoyl}-butyricacid

[0485]2-Amino-4-[2-(2-benzyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid

[0486]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-phenoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0487] Another, more preferred series of compounds includes thefollowing as well as their stereoisomers, tautomers, salts, and mixturesthereof:

[0488] 3-(1H-Benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester

[0489] 3,4-Dihydro-2H-[1,4]thiazine-3,5-dicarboxylic acid 5-ethyl ester

[0490] 3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester

[0491] 3-(5-Nitro-1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0492] 2-oxo-3-(2-thioxo-imidazolidin-1-yl)-propionic acid ethyl ester

[0493] 3-(5-Methoxy-1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0494] 3-(4,5-Dihydro-thiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester

[0495]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0496]2-Amino-N-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethyl]-succinamicacid

[0497]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid

[0498]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(1-methoxycarbonyl-2-phenylethylcarbamoyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid

[0499]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(3-morpholin-4-yl-2,3-dioxo-propylsulfanyl)-ethylcarbamoyl]-butyricacid

[0500]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0501]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0502]2-Amino-4-[2-(2-butoxycarbonyl-2-methoxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid

[0503]2-Amino-4-[2-(2-benzyloxyimino-2-butoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid.

[0504] Presently most preferred (particularly in the practice of themethods of the invention) is the following series of compounds includingtheir stereoisomers, tautomers, salts, and mixtures thereof:

[0505]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-oxo-2-pentyloxycarbonyl-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0506]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0507]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0508]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]butyricacid,

[0509]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]butyricacid,

[0510]2-amino-4-[2-(2-butoxycarbonyl-2-methoxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0511]2-amino-4-[2-(2-benzyloxyimino-2-butoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0512]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0513]2-amino-4-[2-(2-butoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0514]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0515]2-amino-4-[2-(2-benzyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl slat thereof,

[0516]2-amino-4-{-(carboxymethyl-carbamoyl)-2-[2-ethoxycarbonyl-2-(4-nitro-benzyloxyimino)-ethylsulfanyl]-ethylcarbamoyl}-butyricacid,

[0517]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-phenoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0518]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-ethoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid or the di-HCl salt thereof,

[0519]2-amino-4-[2-(2-tert-butoxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the di-HCl salt thereof,

[0520]4-[2-(2-allyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-2-amino-butyricacid or the di-HCl salt thereof,

[0521]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[3-(4-methyl-piperidin-1-yl)-2,3-dioxo-propylsulfanyl]-ethylcarbamoyl}-butyricacid,

[0522]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hydroxyimino-3-oxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyric acid,

[0523]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-diethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0524]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0525]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(1-methoxycarbonyl-2-phenylethylcarbamoyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid,

[0526]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-cyclohexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0527] 2-Amino-4-[2-(2-benzylcarbamoyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyric acid,

[0528]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(3-morpholin4-yl-2,3-dioxo-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0529]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0530]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0531]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-pyrrolidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0532]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-octylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0533]1-{3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionyl}-pyrrolidine-2-carboxylicacid methyl ester,

[0534]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0535]2-{3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionylamino}-3-methyl-pentanoicacid methyl ester,

[0536]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-dimethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0537]2-amino-4-(1-(carboxymethyl-carbamoyl)-2-{2-[2-(4-hydroxy-phenyl)-1-methoxycarbonyl-ethylcarbamoyl]-2-oxo-ethylsulfanyl}-ethylcarbamoyl)-butyricacid,

[0538] 3-(1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid,

[0539] 2-oxo-3-(4-oxo-3,4-dihydro-quinazolin-2-ylsulfanyl)-propionicacid ethyl ester,

[0540] 3-[1-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacid ethyl ester,

[0541] 3-(benzoselenazol-2-ylsulfanyl)-2-oxo-propionic acid ethyl ester,

[0542] 3-(1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0543] 3-(5-chloro-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0544] 3-(5-nitro-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0545] 3-(5-methoxy-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester,

[0546] 3-(4,5-dihydro-thiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0547] 2-hydroxyimino-3-p-tolylsulfanyl-propionic acid methyl ester,

[0548] 2-hydroxyimino-3-p-tolylsulfanyl-propionic acid,

[0549] 2-hydroxyimino-3-p-tolylsulfanyl-propionic acid ethyl ester,

[0550] 3-(5-chloro-benzothiazol-2-ylsulfanyl)-2-hydroxyimino-propionicacid ethyl ester,

[0551]2-hydroxyimino-3-(5-methoxy-1H-benzoimidazol-2-ylsulfanyl)-propionicacid ethyl ester,

[0552] 3-(1H-benzoimidazol-2-ylsulfanyl)-2-hydroxyimino-propionic acidethyl ester,

[0553] 2-hydroxyimino-N-phenyl-3-p-tolylsulfanyl-propionamide, and

[0554] 1-piperidin-1-yl-3-p-tolylsulfanyl-propane-1,2-dione 2-oxime.

Utility, Testing and Administration

[0555] General Utility

[0556] Compounds of the present invention are useful in treating anumber of disorders, particularly those characterized by oxidativestress and/or inflammation. In particular, compounds of the presentinvention can be used in the treatment of ischemia including stroke,cerebral ischemia, myocardial ischemia, retinal ischemia, myocardialinfarction and post-surgical cognitive dysfunction; neurodegenerativedisorders including Alzheimer's, dementia and Parkinson's disease;peripheral neuropathy, including spinal cord injury, head injury andsurgical trauma; inflammatory disorders including diabetes, renaldisease, pre-menstrual syndrome, asthma, cardiopulmonary inflammatorydisorders, heart failure (including chronic and congestive heartfailure), rheumatoid arthritis, osteoarthritis, muscle fatigue andintermittent claudication; and for the preservation of allograft tissueand organs for transplantation.

[0557] Certain of the conditions characterized by oxidative stress fallwithin the group: myocardial ischemia, myocardial infarction,cardiopulmonary inflammatory disorders; and heart failure (includingchronic and congestive heart failure); these are treated particularlywith compounds of Formula I where W is ═O and where Z is —OR. Anothergroup of conditions characterized by oxidative stress includes: stroke,cerebral ischemia, retinal ischemia, post-surgical cognitivedysfunctions (e.g., following bypass surgery), peripheral neuropathyspinal chord injury, head injury and surgical trauma, andneurodegenerative disorders including Alzheimer's, dementia andParkinson's disease; these are treated particularly with compounds ofFormula I where W is ═O or ═N—OR^(a) and where Z is —OR or —NR^(b)R^(c).Another grouping of diseases characterized by oxidative stress andinvolving inflammatory and/or autoimmune components includes: diabetes;renal disease; pre-menstrual syndrome; asthma, rheumatoid arthritis;osteoarthritis, muscle fatigue; and intermittent claudication.

[0558] Testing

[0559] This section describes how compositions incorporatingcompositions of the present invention are selected, using in vitroand/or in vivo animal models, for example, and used as therapeuticinterventions in three exemplary indications, i.e., stroke, chronicheart failure and myocardial infarction.

[0560] Insults to the brain that disrupt its blood supply, as inischemia, or its oxygen supply, as in hypoxia (low oxygen) or anoxia (nooxygen), rapidly cause neuronal imbalance leading to cell death (Flynn,C. J., et al., 1989, in G. Siegel et al., (Eds), Basic Neurochemistry,Raven Press, NY). Investigations into the cellular and molecularmechanisms that lead to neuronal damage and inflammation associated withvarious types of brain ischemia can be carried out using in vitro modelsystems, such as primary cell cultures, that retain the metaboliccharacteristics of neurons in vivo. The use of such cell-based modelshas led to advances in identification of biochemical mechanisms leadingto neuronal death in conditions such as anoxia, hypoglycemia,excitotoxicity, and exposure to reactive oxygen species. Neuronal celllines such as the pheochromocytoma cell line, PC12, are also usefulmodels for studying the effects of oxidative stress on the structure andfunction of neuron-specific proteins that are expressed in the celllines. As many neuronal cell lines do not express all the properties ofgenuine neurons, primary neuronal cultures are now widely used as invitro models in which to discern the processes that occur in intactbrain.

[0561] In vitro models of ischemia approximate oxygen and glucosedeprivation that mimic in vivo conditions, for example, by placingneuronal cultures into large anaerobic or hypoxic chambers andexchanging culture medium with de-oxygenated and defined ioniccomposition media. The toxic overstimulation of neuronal glutamatereceptors, especially N-methyl-D-aspartate (NMDA) receptors, contributesto hypoxic-ischemic neuronal injury (Choi, D. M., 1988, Neuron 1:623-634), ischemic induction of reactive oxygen species (ROS) (Watson,B. D., et al, 1988, Ann NY Acad Sci., 59: 269-281), excessive calciuminflux (Grotta, J. C., 1988, Stroke 19: 447-454), arachidonic acidincrease (Siesjo, B. K., 1981, J. Cereb. Blood Flow Metab. 1: 155-186)and DNA damage (MacManus, J. P., et al., 1993, Neurosci. Lett., 164:89-92), each causing a cascade of neurodegeneration.

[0562] Primary embryonic hippocampal neuronal cells are widelyrecognized as useful in models of neuronal function. The hippocampus isa source of a relatively homogenous population of neurons withwell-characterized properties typical of central nervous system (CNS)neurons in general. Pyramidal neurons, the principal cell type in thehippocampus, have been estimated to account for 85% to 90% of the totalneuronal population (Banker and Goslin, 1998, Culturing Nerve Cells,2^(nd) edition. The MIT Press, Cambridge, Mass.). The hippocampus alsoexhibits a remarkable capacity for activity-dependent changes insynaptic function, such as long-term potentiation (Hawkins RD, Kandel ER, Siegelbaum S A. (1993) Learning to modulate transmitter release:themes and variations in synaptic plasticity [review], Ann. RevNeurosci. 16:625-665.).

[0563] In experiments carried out in support of the present inventionaccording to methods detailed in the Examples, anoxia/ischemia wasinduced in primary cultures of hippocampal neuronal cells, and compoundswere tested for their ability to prevent cell death. Compounds found tohave activity in such in vitro assays are then further tested in one ormore animal models of cerebral ischemia (“stroke”), such as the middlecerebral artery occlusion (MCAO) model in rats.

[0564] Briefly, primary cultures of hippocampal neurons are used to testcompounds for activity in neuronal protection. Hippocampal cultures aretypically prepared from 18- to 19-day fetal rats. At this age, thegeneration of pyramidal neurons, which begins in the rat at about El5,is essentially complete. The brain tissue at this stage is relativelyeasy to dissociate, the meninges are removed readily, and the number ofglial cells still is relatively modest (Park L C, Calingasan N Y, UchidaK, Zhang H, Gibson G E. (2000) Metabolic impairment elicits brain celltype-selective changes in oxidative stress and cell death in culture. JNeurochem 74(1):114-124).

[0565] In order to evaluate the activity of compounds of the presentinvention, a test compound is assessed for its ability to protect cellsagainst one or more standard stressors, including hypoxia, as detailedin the Examples. In general, desirable therapeutic compound candidatesare effective in this model at concentrations less than about 10 mM,more preferably at concentrations, less than about 1 mM and even morepreferably, less than about 100 μM. By effective, it is meant that suchcompounds protect at least 20%, preferably 30%, more preferably 40% andeven more preferably 50% or more of the cells tested fromstressor-induced death. By way of example, compounds that are effectivein providing protection over a concentration a range of about 1 to 1000μM would be expected to provide neuroprotection in vivo. Since precisevalues may vary depending upon the specific conditions under which theneuroprotective cell assay is carried out, it is the intent of thepresent disclosure to provide the foregoing criteria as guidance in theform of a benchmark against which to compare subsequently testedcompounds, rather than to provide absolute concentrations at which thecompounds of the present invention are considered to be effective.Typically, compounds that are found to be neuroprotective in such invitro cell systems are then further tested in an in vivo animal model ofneuroprotection, such as the rat middle cerebral artery occlusion modeldescribed below, or other appropriate models such as are well known inthe art.

[0566] Cerebral ischemic insults are modeled in animals by occludingvessels to, or within, the cranium (Molinari, G. F., 1986, in H. J. M.Barnett, et al., (Eds) Stroke: Pathophysiology, Diagnosis andManagement, Vol. 1, Churchill Livingstone, N.Y.). The rat middlecerebral artery occlusion (MCAO) model is one of the most widely usedtechniques to induce transient focal cerebral ischemia approximatingcerebral ischemic damage in humans, e.g., those who suffer from astroke. The middle cerebral artery used as the ischemic trigger in thismodel is the most affected vessel in human stroke. The modelalso entailsa period of reperfusion, which typically occurs in human stroke victims.MCAO involving a two-hour occlusion has been found to produce themaximum size of cortical infarction obtainable without increasedmortality at twenty-four hours.

[0567] Briefly, a nylon filament is implanted into the right carotidartery of the rat. To effect occlusion, the rat is anesthetized, and thefilament is advanced into the internal carotid artery 18-20 mm from thepoint of bifurcation of internal and external arteries and a suture istightly ligated around the filament for a period of two hours. Two hourspost occlusion, animals are re-anesthetized, and the filament isremoved, to allow reperfusion for the remainder of the experiment. Testdrugs can be administered any time during this process—before, during orafter occlusion, and can be administered by one or more of a variety ofmeans, including but not limited to intracerebroventricular (ICV)infusion, intravenous (IV) infusion, intraperitoneal (IP)administration, as well as enteral administration (e.g., gavage).Animals are maintained normothermic during the experiment, as describedin the Examples. At a pre-determined time following occlusion andreperfusion, animals are sacrificed and their brains are removed andprocessed for assessment of damage as measured by infarct volume. Ingeneral, compounds are considered to have activity in this model, ifthey provide a significant reduction in total infarct volume at a dosethat is less than about 10 mg/kg, preferably less than 1 mg/kg, morepreferably less than 100 μg/kg and even more preferably less than about1 μg/kg, when administered ICV or IV. By significant reduction of totalinfarct volume is meant a reduction of at least 20%, preferably at least30%, more preferably at least 40%, and even more preferably about 50%,compared to control values.

[0568] Further validation of efficacy in neuroprotection can be assessedin functional tests, such as the grip strength test or the rotorod test.Animals treated with compounds that show neuroprotection maintain theirpre-MCAO grip strength values after MCAO, as compared to untreatedanimals, who showed a significant reduction in grip strength, indicatingloss of sensorimotor function. Likewise, animals treated with compoundsthat show neuroprotection also maintained their pre-MCAO rotorodactivity scores after MCAO, as compared to untreated animals, who showeda significant reduction in rotorod scores, indicating loss ofsensorimotor function at higher brain levels.

[0569] Similarly, primary cultures of myocytes can be used to testcompounds in vitro for ability to provide protection against heartdamage, resulting for example from myocardial ischemia or congestiveheart failure. Preparation of myocardiocytes from neonatal rats isdescribed in the Examples. Such cells are typically used to studymolecular models of myocardial ischemia (Webster, K A, Discher, D J &Bishopric, N H. 1995. J. Mol. Cell Cardiol. 27:453-458; Camilleri, L,Moins, N, Papon, J, Maublant, J, Bailly, P, de Riberolles, C & Veyre, A.1997. Cell Biol. & Toxicol. 13:435-444; Bielawska, A E, Shapiro, J P,Jiang, L, Melkonyan, H S, Piot, C, Wolfe, C L, Tomei, L D, Hannun, Y A &Umansky, S R. 1997. Am. J. PathoL 151:1257-1263) and are thereforeaccepted as indicative of myoprotective activity. Exemplary stressorassays for this purpose are provided in the Examples. For example,cardiomyocytes in culture exhibit contractile (“beating”) activity; eachcardiomyocyte contraction is associated with a rise in intracellularcalcium termed a “calcium transient”. These calcium transients can bemeasured using Fluo-4, a fluorescent dye which exhibits largefluorescence intensity increases upon the binding of calcium. This assayis cell-based and tests the ability of potential cytoprotectantmolecules to guard against ischemic damage and allow the cells tomaintain their contractile function.

[0570] Further validation of compounds can be carried out in a wholeorgan assay, such as the isolated heart model of cardiac function.Similarly, compounds can be further validated in additional animalmodels of disease (e.g., diabetes, renal failure, asthma, musclefatigue, inflammation), such as are well known in the art.

[0571] Administration

[0572] The compounds of Formula I are administered at a therapeuticallyeffective dosage, e.g., a dosage sufficient to provide treatment for thedisease states previously described. Administration of the compounds ofthe invention or the pharmaceutically acceptable salts thereof can bevia any of the accepted modes of administration for agents that servesimilar utilities.

[0573] While human dosage levels have yet to be optimized for thecompounds of the invention, generally, a daily dose is from about 0.01to 2.0 mg/kg of body weight, preferably about 0.1 to 1.5 mg/kg of bodyweight, and most preferably about 0.3 to 1.0 mg/kg of body weight. Thus,for administration to a 70 kg person, the dosage range would be about0.7 to 140 mg per day, preferably about 7.0 to 105 mg per day, and mostpreferably about 21 to 70 mg per day. Administration can be as a singledose (e.g., as a bolus) or as an initial bolus followed by continuousinfusion of the remaining portion of a complete dose over time, e.g., 1to 7 days. The amount of active compound administered will, of course,be dependent on the subject and disease state being treated, theseverity of the affliction, the manner and schedule of administrationand the judgment of the prescribing physician.

[0574] In employing the compounds of this invention for treatment of theabove conditions, any pharmaceutically acceptable mode of administrationcan be used. The compounds of Formula I can be administered either aloneor in combination with other pharmaceutically acceptable excipients,including solid, semi-solid, liquid or aerosol dosage forms, such as,for example, tablets, capsules, powders, liquids, suspensions,suppositories, aerosols or the like. The compounds of Formula I can alsobe administered in sustained or controlled release dosage forms,including depot injections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for the prolongedadministration of the compound at a predetermined rate, preferably inunit dosage forms suitable for single administration of precise dosages.The compositions will typically include a conventional pharmaceuticalcarrier or excipient and a compound of Formula I or a pharmaceuticallyacceptable salt thereof. In addition, these compositions may includeother medicinal agents, pharmaceutical agents, carriers, adjuvants, andthe like, including, but not limited to anticoagulants, blood clotdissolvers, permeability enhancers and slow release formulations.

[0575] Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,preferably about 0.5% to 50%, by weight of a compound or salt of FormulaI, the remainder being suitable pharmaceutical excipients, carriers,etc.

[0576] One preferred manner of administration for the conditionsdetailed above is oral, using a convenient daily dosage regimen, whichcan be adjusted according to the degree of affliction. For such oraladministration, a pharmaceutically acceptable, non-toxic composition isformed by the incorporation of any of the normally employed excipients,such as, for example, mannitol, lactose, starch, magnesium stearate,sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose,gelatin, sucrose, magnesium carbonate, and the like. Such compositionstake the form of solutions, suspensions, tablets, dispersible tablets,pills, capsules, powders, sustained release formulations and the like.

[0577] Preferably the compositions will take the form of a pill ortablet and thus the composition will contain, along with the activeingredient, a diluent such as lactose, sucrose, dicalcium phosphate, orthe like; a lubricant such as magnesium stearate or the like; and abinder such as starch, gum acacia, polyvinylpyrrolidine, gelatin,cellulose and derivatives thereof, and the like.

[0578] Liquid pharmaceutically administrable compositions can, forexample, be prepared by dissolving, dispersing, etc. an active compoundas defined above and optional pharmaceutical adjuvants in a carrier,such as, for example, water, saline, aqueous dextrose, glycerol,glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, or solubilizingagents, pH buffering agents and the like, for example, sodium acetate,sodium citrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine acetate, triethanolamine oleate, etc. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. Thecomposition or formulation to be administered will, in any event,contain a quantity of the active compound in an amount effective toalleviate the symptoms of the subject being treated.

[0579] Dosage forms or compositions containing active ingredient in therange of 0.005% to 95% with the balance made up from non-toxic carriermay be prepared.

[0580] For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, talcum, cellulosederivatives, sodium crosscarmellose, glucose, sucrose, magnesiumcarbonate, sodium saccharin, talcum and the like. Such compositions takethe form of solutions, suspensions, tablets, capsules, powders,sustained release formulations and the like. Such compositions maycontain 0.01%-95% active ingredient, preferably 0.1-50%.

[0581] For a solid dosage form, the solution or suspension, in forexample propylene carbonate, vegetable oils or triglycerides, ispreferably encapsulated in a gelatin capsule. Such diester solutions,and the preparation and encapsulation thereof, are disclosed in U.S.Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form,the solution, e.g. in a polyethylene glycol, may be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g. water, to be easily measured for administration.

[0582] Alternatively, liquid or semi-solid oral formulations may beprepared by dissolving or dispersing the active compound or salt invegetable oils, glycols, triglycerides, propylene glycol esters (e.g.propylene carbonate) and the like, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

[0583] Other useful formulations include those set forth in U.S. Pat.Nos. Re. 28,819 and 4,358,603.

[0584] The formulation can be administered in a single unit dosage formfor continuous treatment or in a single unit dosage form ad libitum whenrelief of symptoms is specifically required. For example, theformulation may be administered as a bolus or as a continuousintravenous infustion after onset of symptoms of stroke, myocardialinfarction or chronic heart failure.

[0585] Parenteral administration is generally characterized byinjection, either subcutaneously, intramuscularly or intravenously.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,solubility enhancers, and the like, such as for example, sodium acetate,sorbitan monolaurate, triethanolamine oleate, cyclodextrins, etc.

[0586] A more recently devised approach for parenteral administrationemploys the implantation of a slow-release or sustained-release system,such that a constant level of dosage is maintained. See, e.g., U.S. Pat.No. 3,710,795. The percentage of active compound contained in suchparenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject. However, percentages of active ingredient of 0.01% to 10% insolution are employable, and will be higher if the composition is asolid, which will be subsequently diluted to the above percentages.Preferably the composition will comprise 0.2-2% of the active agent insolution.

[0587] Nasal solutions of the active compound alone or in combinationwith other pharmaceutically acceptable excipients can also beadministered.

[0588] Formulations of the active compound or a salt may also beadministered to the respiratory tract as an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation have diameters of less than 50 microns,preferably less than 10 microns.

EXAMPLES

[0589] The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

[0590] General Characterization Methods

[0591] As reported in most cases of the following examples, NuclearMagnetic Resonance (NMR) spectra were recorded on a Bruker Avance 300spectrometer using tetramethyl silane (TMS) as the internalreference;mass spectra were obtained on an Agilent 110 LC/MSD instrumentusing either electrospray ionization (positive or negative mode) (ESI)or atmospheric pressure chemical ionization (positive or negative mode)(APCI).

Example 1 Formula Ia where A is 1H-Benzimidazole-2-yl, R is Ethyl, and Xis S

[0592] A solution of 2-mercaptobenzimidazole (200 mg, 1.33 mmol) andethyl 3-bromopyruvate (0.20 mL, 1.43 mmol) in methanol (2.5 mL) andacetone (2 mL) was shaken at 20° C. for 4 hours. The solvents wereremoved under the reduced pressure on a rotary evaporator. The residuewas triturated with ethyl acetate. Solvent was decanted and the residuewas dissolved in methylene chloride. The solution was washed withdiluted aqueous sodium bicarbonate solution and water, and then driedover magnesium sulfate. Removal of the solvent gave the expectedproduct, 3-(1H-Benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester (326 mg, 93%). ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.31 (br. s, 1H),7.24 (br. s, 1H), 7.15-7.05 (m, 2H), 4.32 (q, J=7.1 Hz, 2H), 4.29 (br.s, 1H), 3.90 (br. s, 1H), 1.24 (t, J=7.1 Hz, 3H). MS (ESI): m/z 265(M+1, 100).

Example 2 Formula Ia where A is 5-Methoxy-1H-benzimidazole-2-yl, R isEthyl, and X is S

[0593] A solution of 2-mercapto-5-methylbenzimidazole (200 mg, 1.22mmol) and ethyl 3-bromopyruvate (0.20 mL, 1.43 mmol) in methanol (2 mL)and acetone (3 mL) was shaken at room temperature for 4 hours. Thesolvents were removed under the reduced pressure on a rotary evaporator.The residue was triturated with ethyl acetate. Solvent was decanted andthe residue was dissolved in methylene chloride. The solution was washedwith diluted aqueous sodium bicarbonate solution and water, and thendried over magnesium sulfate. Removal of the solvent gave the expectedproduct, 3-(5-methoxy-1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionicacid ethyl ester (300 mg, 88%). ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 7.19(br. s, 1H), 7.10 and 7.04 (br. 2 s, 1H), 6.94 (d, J=8.2 Hz, 1H), 4.34(q, J=7.1 Hz, 2H), 4.31 (br. s, 1H), 3.91 (br. s, 1H), 2.37 (br. s, 3H),1.26 (t, J=7.1 Hz, 3H). MS (ESI): m/z 279 (M+1, 100).

Example 3 Formula Ia where A is 5-Methyl-1H-benzimidazole-2-yl, R isEthyl, and X is S

[0594] A solution of 5-methoxy-2-benzimidazolethiol (200 mg, 1.11 mmol)and ethyl 3-bromopyruvate (0.20 mL, 1.43 mmol) in methanol (5 mL) andacetone (2 mL) was shaken at room temperature for 4 hours. The solventswere removed under the reduced pressure on a rotary evaporator. Theresidue was triturated with ethyl acetate. Solvent was decanted and theresidue was dissolved in methylene chloride. The solution was washedwith diluted aqueous sodium bicarbonate solution and water, and thendried over magnesium sulfate. Removal of the solvent gave the expectedproduct, 3-(5-Methyl-1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester (300 mg, 92%). ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 7.19 (br. s,1H), 6.75 (br. s, 1H), 6.69 (dd, J=2.3 Hz, J=8.8 Hz, 1H), 4.30 (q, J=7.1Hz, 2H), 4.29 (br. s, 1H), 3.93 (br. s, 1H), 3.69 (s, 3H), 1.24 (t,J=7.1 Hz, 3H). MS (ESI): m/z 295 (M+1, 100).

Example 4 Preparation of 3,4-dihydro-2H-[1,4]thiazine-3,5-dicarboxylicacid

[0595] To a solution of 1-cysteine (1.00 g, 8.25 mmol) in water (20 mL)was added ethyl 3-bromopyruvate (1.61 g, 8.25 mmol). The pH of solutionwas adjusted to about 4 with aqueous sodium bicarbonate solution. Afterstirred at room temperature for 30 minutes, the mixture was washed withmethylene chloride. The aqueous phase was separated and then evaporatedto dryness under lyophilization conditions. The methanol solution of theobtained residue was passed through a silica gel pack, resulting in 470mg of pure product (32%). ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 6.16 (d,J=1.2 Hz, 1H), 4.23 (d, J=7.1 Hz, 2H), 3.86 (dd, J=2.7 Hz, J=8.2 Hz,2H), 3.25-3.15 (m, 1H), 2.78 (dd, J=8.2 Hz, J=12.2 Hz, 1H), 1.30 (t,J=7.1 Hz, 3H). ¹³CNMR (75 Hz, D₃COD): δ (ppm): 177.4, 164.6, 129.9,103.0, 62.3, 56.7, 28.8, 14.7.

Example 5 Formula Ia where A is4-[2-(3,4-Dihydroxy-phenyl)-vinyl]-pyridinium bromide, R is H, and X isa Covalent Bond

[0596] 4-Bromomethyl-1,2-bis-methoxymethoxy-benzene

[0597] A suspension of triphenyl phosphine (262 mg) in acetonitrile (20mL) was cooled on an ice-water bath. Bromine (160 mg) was added to thissuspension through a syringe with stirring. The color of bromine soondisappeared. The solution was stirred at 0° C. for 10 more minutes andthen N,N-diisopropyl ethylamine (2.2 eq. ca 360 μL) and(3,4-bis-methoxymethoxy-phenyl)-methanol (228 mg, 1 eq. either neat orin CH₃CN solution) were introduced at 0° C. The resulted solution wasslightly basic. It was stirred at 0° C. for 30 min before diluted withethyl ether. After washed with saturated NaH₂PO₄ solution, dried overMgSO₄, and evaporated to dryness, it gave rise to a solid residue.Purification by silica gel column chromatography (eluting with ethylacetate:hexane 3:7) rendered 180 mg of thick oil (yield 62%).

[0598] (3,4-Bis-methoxymethoxy-benzyl)-triphenVl-phosphonium bromide

[0599] A solution of 4-bromomethyl-1,2-bis-methoxymethoxy-benzene (1eq)and PPh₃ (1.02 eq) in toluene was refluxed (oil bath 100-130° C.)overnight. White precipitates were collected and washed with cooltoluene. The solid was dried under vacuum and the yield was about 90%.

[0600] 4-[2-(3 4-Bis-methoxymethoxy-phenyl)-vinyl]-pyridine

[0601] To a mixture of(3,4-Bis-methoxymethoxy-benzyl)-triphenyl-phosphonium bromide (800 mg)and 4-formylpyridine (130 mg) in ethanol was added lithium ethoxidesolution (1.5 mL, 1.0 M in ethanol) over a period of 130 minutes. Afteradditional 30 minutes of stirring, the solvent was stripped off byrotary evaporation under diminished pressure. The residue was dissolvedin water and extracted with ethyl acetate. Back washed with water, theorganic solution was dried over magnesium sulfate and concentrated on arotary evaporator. Purification was carried out on a silica gel columnusing gradient ethyl acetate in dichloromethane (5-20%). It led to asemi-solid mixture of E- and Z-isomers in ca 80% yield.

[0602]4-[2-(3,4-Dihydroxy-phenyl)-vinyl]-1-(2-ethoxycarbonyl-2-oxo-ethyl)-pyridinium;bromide

[0603] A solution of4-[2-(3,4-bis-methoxymethoxy-phenyl)-vinyl]-pyridine (70 mg) and ethyl3-bromopyruvate (200 μL) in 1, 4-dioxane (10 mL) was stirred at 90-110°C. overnight. The initial colorless solution turned into brown and someorange precipitate appeared. The dioxane solvent was stripped off on arotary evaporator and the resulted solid was washed extensively withethyl ether. Without further purification, it was dissolved in methanol(20 mL) and concentrate HBr (48% aqueous solution, 10 drops) wasintroduced. After stirred at room temperature overnight, followed byremoval of solvent by rotary evaporation, the residue was successivelywashed with ethyl ether, ethyl acetate, dichloromethane, and then driedunder vacuum. This gave the expected product,4-[2-(3,4-dihydroxy-phenyl)-vinyl]-1-(2-ethoxycarbonyl-2-oxo-ethyl)-pyridinium;bromide, as an orange solid (83 mg, 87%). ¹H-NMR (300 MHz, DMSO-d₆) δ(ppm): 8.83 (dd, J=1.8, 7.1 Hz), 8.76-8.55 (m), 8.22 (dd, J=1.5, 7.0Hz), 8.10-8.00 (m), 7.80 (dd, J=6.2, 16.1 Hz), 7.75-7.5 (m), 7.31-7.10(m), 6.87 (d, J=8.2 Hz), 4.72 (q, J=13.5 Hz), 4.34-4.26 (m), 3.86-3.53(m), 1.52-1.15 (m). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 167.3,155.7,154.8,148.4, 148.2, 145.3,145.2, 144.9, 142.5, 141.8, 139.9,131.3,131.2,128.3,128.1,126.8, 126.7,122.1, 122.0, 121.7, 121.6, 119.0, 118.6,115.0,114.9, 114.0, 113.7,113.6, 94.4, 63.5, 62.0, 61.7,12.6, and 12.4.

Example 6 3-(4,5-Dihydro-1H-imidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester

[0604] 6A. Formula Ia where A is 4,5-Dihydro-1H-imidazol-2-yl, R isEthyl, and X is S

[0605] A mixture of 2-imidazolidinethione (1.02 g, 10 mmol), ethyl3-bromopyruvate (1.95 g, 10 mmol), potassium carbonate (1.38 g, 10mmol), sodium iodide (10 mg), Adogen (200 mg) in N,N-dimethyl formamide(15 mL) was stirred at 20° C. for 15 hours under nitrogen. The reactionmixture was diluted with water and then extracted ethyl acetate. Organicphase was dried over sodium sulfate, evaporated, and chromatographed(silica gel, dichloromethane-methanol 9:1), giving the expected product,3-(4,5-dihydro-1H-imidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester, as a brown solid (1.8 g, 83%). ¹H-NMR (CDCl₃, 300 MHz) δ (ppm):4.36 (d, J=7.1 Hz, 2H), 4.25-4.00 (m, 3H), 3.57 (d, J=11.2 Hz, 1H),3.50-3.35 (m, 1H), 3.25-3.10 (m, 1H), 1.37 (t, J=7.1 Hz, 3H).

[0606] 6B. Other Compounds of Formula Ia

[0607] Similarly, by following the procedures of Example 6A andsubstituting 2-imidazolidinethione with2-amino-3-(1H-imidazol-4-yl)propionic acid, there is obtained2-amino-3-[1-(2-ethoxycarbonyl-2-oxo-ethyl)-1H-imidazol-4-yl]-propionicacid.

Example 7 2-Hydroxy-3-(1H-imidazol-2-ylsulfanyl)-5-oxo-hex-2-enedioicacid diethyl ester

[0608] A solution of 3H-Imidazole-4-thiol (0.1 g, 1.0 mmol), ethyl3-bromopyruvate (0.31 g, 1.6 mmol), and triethylamine (0.2 mL) inMeOH/CH₂Cl₂ (1.5 mL/1.5 mL) in a capped vial was shaken at roomtemperature overnight. After removal of the volatiles, the residues weredissolved in methylene chloride and loaded onto the top of a silica gelcolumn. Elution with ethyl acetate in methylene chloride (10%) gave theproduct (a di-pyruvate conjugate) as a semi solid (87 mg, 35%). ¹H-NMR(CDCl₃, 300 MHz) δ (ppm): 1.19 (t, 3H), 1.37 (t, 3H), 4.12 (q, 2H), 4.37(q, 2H), 4.59 (d, 1H), 4.68 (d, 1H), 7.06 (ss, 2H).

Example 83-[2-Amino-9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-9H-purin-6-ylsulfanyl]-2-oxo-propionicacid ethyl ester

[0609] 8A. Formula Ia where A is3-[2-Amino-9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-9H-purin-6-yl,R is Ethyl, and X is S

[0610] To a solution of (−)-2-amino-6-mercaptopurine riboside (100 mg,0.33 mmol) in N,N-dimethyl formamide (2 mL) was added ethyl3-bromopyruvate (0.050 mL, 0.37 mmol) under nitrogen. After stirred at20° C. for 1 hour, the reaction mixture was diluted with water and thenconcentrated on a rotary evaporator under vacuum. The residue wastriturated with ethyl acetate. The ethyl acetate phase was concentratedand chromatographed (silica gel, methylene chloride-methanol 100:5 to100:10), affording 21 mg of the expected product,3-[2-amino-9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-9H-purin-6-ylsulfanyl]-2-oxo-propionicacid ethyl ester. ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 8.13 (s, 1H), 5.87(d, J=6.1 Hz, 1H), 4.70-4.60 (m, 1H), 4.35-4.30 (m, 2H), 4.20-4.05 (m,4H), 3.90-3.70 (m, 2H), 1.20 (t, J=7.1 Hz, 3H). MS (ESI) m/z: 414 (M+1,100), 432 (M+H₂O+1, 84), 446 (M+Na, 45).

[0611] 8B. Other Compounds of Formula Ia

[0612] Similarly, by following the procedures of Example 8A andsubstituting (−)-2-amino-6-mercaptopurine riboside with the following:

[0613] (−)-6-mercaptopurine, and

[0614] (−)-6-mercaptopurine riboside

[0615] there are obtained the following:

[0616] 2-oxo-3-(9H-purin-6-ylsulfanyl)-propionic acid ethyl ester, and

[0617]3-[9-(3,4-Dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-9H-purin-6-ylsulfanyl]-2-oxo-propionicacid ethyl ester.

Example 9 2-Oxo-3-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-propionic acidethyl ester sodium salt

[0618] 9A. Formula Ia where A is 5-sulfo-1H-benzoimidazol-2-yl sodiumsalt, R is Ethyl, and X is S

[0619] To a solution of 2-mercapto-5-benzimidazole-sulfonic acid sodiumsalt (252 mg, 1.0 mmol) in water (3 mL) was added ethyl 3-bromopyruvate(0.150 mL, 1.19 mmol) under nitrogen. The resulted solution was stirredat 20° C. for 1 hour. Water was removed under vacuum and the residuewashed with ethyl acetate. Dried over sodium sulfate, the organicsolution was evaporated to dryness under vacuum. This gave 300 mg (82%)of product, 2-oxo-3-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-propionicacid ethyl ester sodium salt, which was sufficiently pure to be usedwithout further purification. ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 8.70 (dd,J=0.6 Hz, J=1.5 Hz, 1H), 7.95 (dd, J=1.5 Hz, J=8.6 Hz, 1H), 7.74 (dd,J=0.6 Hz, J=1.5 Hz, 1H), 4.27 (q, J=7.1 Hz, 2H), 3.93 (d, J=14.2 Hz,1H), 3.81 (d, J=14.2 Hz, 1H), 1.30 (t, J=7.1 Hz, 3H). MS (ESI), 367(M+1, 28), 345 (M−Na+1, 100).

[0620] 9B. Other Compounds of Formula Ia

[0621] Similarly, by following the procedures of Example 9A andsubstituting 2-mercapto-5-benzimidazole-sulfonic acid sodium salt with6-ethoxy-2-mercaptobenzimidazole, there is obtained3-(6-ethoxy-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethyl ester.

Example 10 Formula Ia where A is 5-Amino-2H-[1,2,4] triazol-3-yl, R isEthyl, and X is S

[0622] To a suspension of 3-amino-5-mercapto-1,2,4-triazole (116 mg, 1.0mmol) in methanol (3 mL) was added ethyl 3-bromopyruvate (0.150 mL, 1.19mmol) under nitrogen. The resulting solution was stirred at 20° C. for 1hour. A clear solution was formed after about 30 min. Methanol wasremoved under vacuum, leading to 227 mg of solid (99% of yield). ProtonNMR indicated that 3-(5-amino-2H-[1,2,4]triazol-3-ylsulfanyl)-2-oxo-propioic acid ethyl ester was one of themajor products. ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 4.30-4.20 (m, 2H), 3.51(br. s, 2H), 1.30 (t, J=7.1 Hz, 3H).

Example 11

[0623] 11A. Formula Ia where A is 5-Amino-[13,4] thiadiazol-2-yl, R isEthyl, and X is S

[0624] To a suspension of 5-amino-1,3,4-thiadiazole-2-thiol (133 mg, 1.0mmol) in methanol (3 mL) was added ethyl 3-bromopyruvate (0.150 mL, 1.19mmol) under nitrogen. The resulting solution was stirred at 20° C. for 1hour. A clear solution was formed after about 45 min. Methanol wasremoved under vacuum, giving 200 mg of solid (in 81% yield). Proton NMRindicated that 3-(5-amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-oxo-propionic acid ethyl ester was one ofmajor products. ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 4.35-4.20 (m, 2H), 3.58(dd, AB system, 2H), 1.31 (t, J=7.1 Hz, 3H).

[0625] 11 B. Other Compounds of Formula Ia

[0626] Similarly, by following the procedures of Example 11A andsubstituting 5-amino-1,3,4-thiadiazole-2-thiol with5-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-[1,3,4]thiadiazol-2-thiol,there is obtained3-[5-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-[1,3,4]thiadiazol-2-ylsulfanyl]-2-oxo-propionicacid ethyl ester.

Example 12

[0627] 12A. Formula Ia where A is 5-Nitro-1H-benzoimidazol-2-yl, R isEthyl, and X is S

[0628] To a solution of 2-mercapto-5-nitrobenzimidazole (195 mg, 1.0mmol) and ethyl 3-bromopyruvate (0.150 mL, 1.19 mmol) in methanol (2.5mL) and acetone (2 mL) was added imidazole (68 mg, 1.0 mmol). Theresulted solution was shaken at 20° C. for 4 hour. After the removal ofsolvents under vacuum, the residue was triturated with ethyl ether. Theether phase was discarded. The residue was treated with sodiumbicarbonate aqueous solution and ethyl ether. The ether phase was washedsuccessively with sodium bicarbonate aqueous solution, water, and driedover magnesium sulfate. Evaporation to dryness under vacuum gave 200 mg(65% of yield) of the expected product,3-(5-nitro-1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester. Proton NMR showed the product was sufficiently pure to be usedwithout further purification. ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 8.35 and8.08 (2 br. s, 1H), 8.12 (d, J=8.7 Hz, 1H), 7.50 and 7.28 (2 br. s, 1H),4.42 (q, J=7.1 Hz, 2H), 4.41 (br. s, 1H), 4.06 (br. s, 1H), 1.30 (t,J=7.1 Hz, 3H). MS (ESI) m/z: 310 (M+1,100).

[0629] 12B. Other Compounds of Formula Ia

[0630] Similarly, by following the procedures of Example 12A andsubstituting 2-mercapto-5-nitrobenzimidazole with the following:

[0631] 2-amino-3-mercapto propionic acid

[0632] 2-amino-3-sulfinyl propionic acid,

[0633] 2-amino-3-sulfonyl propionic acid, and

[0634] 2-acetylamino-3-mercapto propionic acid

[0635] there are obtained the following:

[0636] 2-amino-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionic acidand 3-(2-amino-2-carboxy-ethylsulfanyl)-2-hydroxy-acrylic acid ethylester,

[0637] 2-amino-3-(2-ethoxycarbonyl-2-oxo-ethanesulfinyl)-propionic acid

[0638] 2-amino-3-(2-ethoxycarbonyl-2-oxo-ethanesulfonyl)-propionic acid,and

[0639] 2-acetylamino-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionicacid.

Example 13

[0640] 13A. Formula Ia where A is 5-Phenyl-[1,3,4] oxadiazol-2-yl, R isEthyl, and X is S

[0641] To a solution of 5-phenyl-1,3,4-oxadiazole-2-thiol (178 mg, 1.0mmol) and ethyl 3-bromopyruvate (0.150 mL, 1.19 mmol) in methanol (2 mL)and acetone (2 mL) was added imidazole (68 mg, 1.0 mmol). The resultedsolution was shaken at 20° C. for 4 hour. After the removal of solventsunder vacuum, the residue was triturated with ethyl ether. The etherphase was discarded. The residue was treated with sodium bicarbonateaqueous solution and ethyl ether. The ether phase was washedsuccessively with sodium bicarbonate aqueous solution, water, and driedover magnesium sulfate. Evaporation to dryness under vacuum gave 209 mg(72% of yield) of the expected product,2-oxo-3-(5-phenyl-[1,3,4]oxadiazol-2-ylsulfanyl)-propionic acid ethylester. Proton NMR showed the product was sufficiently pure to be usedwithout further purification. ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.90-7.80(m, 2H), 7.60-7.35 (m, 3H), 4.41 (q, J=7.1 Hz, 2H), 3.44 (br. s, 2H),1.26 (t, J=7.1 Hz, 3H).

[0642] 13B. Other Compounds of Formula Ia

[0643] Similarly, by following the procedures of Example 13A andsubstituting 5-phenyl-1,3,4-oxadiazole-2-thiol with3-phenyl-1,2,4-oxadiazole-2-thiol, there is obtained2-oxo-3-(3-phenyl-[1,2,4]oxadiazol-5-ylsulfanyl)-propionic acid ethylester.

Example 14 Formula III where R^(3.1) to R^(3.4) are H, and R^(3.5) isCOOH

[0644] To a solution of L-cysteine (2.42 g, 20 mmol) in water (70 mL)was added 3-bromopyruvic acid (3.34 g, 20 mmol) at room temperature withstirring. The clear solution turned cloudy gradually. After stirring for2 hours at room temperature, the white precipitates were filtered,washed with water, and dried under vacuum. This gave a compound ofFormula Ia where A is cysteine, which cyclizes with the enol of pyruvateto afford the title compound of Formula II, 3,4-dihydro-2H-[1,4]thiazine-3,5-dicarboxylic acid, as a grey powder product (2.42 g, 58%).¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 6.00 (s, 1H), 5.17 (br., s, 1H), 4.25(t, 1H), 2.94-3.05 (m, 2H). ¹³C-NMR (DMSO-d₆, 75 MHz) δ (ppm): 25.8,52.3, 98.0,128.8, 163.6, and 172.0. MS (ESI) m/z: 190 (M+H, 100).

Example 15 Formula Ia where A isγ-Glu-Cys-Gly, R is H, and X is aCovalent Bond and Formula II where R¹ is COOH, R² is H, R³ isCH₂—S—Pyruvate, R⁴ is H, R⁵ is H, k is 0, m is 2, and n is 1

[0645] 15A.

[0646] To a solution of glutathione(2-amino-4-[1-(carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-butyricacid) (3.0 g, 9.76 mmol) in water (34 mL) and methanol (4 mL) (degassedand purged with nitrogen) was added 3-bromopyruvic acid (1.63 g, 9.76mmol) at room temperature. After stirring for 3 hours at roomtemperature, the mixture was concentrated on a rotary evaporator underthe reduced pressure. The solution was then washed with methylenechloride thoroughly. The organic layer was discarded. The aqueous layerwas evaporated to dryness under reduced pressure. After drying underhigh vacuum for 48 hours, the expected product was afforded as ayellowish solid in quantitative yield. NMR data indicate that thereexist two tautomeric isomers in the product, namely the keto form,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid, and the enol form,3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacid. ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 6.45 (s, 0.4H), 6.43-6.48 (m,1H), 4.06-4.10 (m, 1H), 3.95 (s, 2H), 2.80-2.93 (m, 2.6H), 2.60-2.65 (m,2H), and 2.14-2.27 (m, 2H).

[0647] 15B. Other Compounds of Formula Ia

[0648] Similarly, by following the procedures of Example 15A andsubstituting glutathione with cysteine, there are obtained3-(2-amino-2-carboxy-ethylsulfanyl)-2-oxo-propionic acid and3-(2-amino-2-carboxy-ethylsulfanyl)-2-hydroxy-acrylic acid.

Example 16 Formula Ia where A is γ-Glu-Cys-Gly, R is Ethyl, and X is aCovalent Bond and Formula II where R¹ is COOH, R² is H, R³ isCH₂—S—Pyruvate Ethyl Ester, R⁴ is H, R⁵ is H, k is 0, mis 2, and n is 1

[0649] To a solution of glutathione(2-amino-4-[1-(carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-butyricacid) (8.14 g, 26.5 mmol) in water (50 mL) and methanol (10 mL)(degassed and purged with nitrogen) was added bromo-3-ethylpyruvate(5.17 g, 26.5 mmol) at room temperature. Upon the addition ofbromo-3-ethylpyruvate, the cloudy suspension turned to yellowishtranslucent almost instantly. After stirring for 2 hours at roomtemperature, the mixture was concentrated on a rotary evaporator underthe reduced pressure. The solution was then washed with methylenechloride thoroughly. The organic layer was discarded. The aqueous layerwas evaporated to dryness under reduced pressure. After drying underhigh vacuum for 48 hours, a white solid was obtained as the product (g,%). NMR data indicate that there exist two tautomeric forms of theproduct, namely the keto form,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid ethyl ester, and the enol form,3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacid ethyl ester. ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 1.17 (t, 3H), 1.93(m, 2H), 2.11 (m, 2H), 2.75-3.28 (m, 2H), 2.29 (ss, 1.6H), 3.93 (s, 2H),4.09 (m, 1H), 4.25 (q, 2H), 4.83 (m, 1H), 6.43 (s, 0.4H). ¹³C-NMR(D₃COD, 75 MHz) δ (ppm): 14.1, 26.6, 32.1, 35.6,40.0,41.4, 53.2,54.6,62.1, 62.7, 100.0, 113.4, 139.7,163.4,170.8,172.3,174.1. MS (ESI)m/z: 422 (M+H, 100), 440 (M+H+H₂O, 42).

Example 17 Formula II where R¹ is COOCH₃, R² is H, R³ is CH₂—S—keto-Pyruvate Ethyl Ester, R⁴ is H, R⁵ is CH₃, k is 0, m is 2, and n is1

[0650] A mixture of2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid (200 mg) and p-toluenesulfonic acid (30 mg) in methanol (100 mL)was heated to reflux for 72 h. The solvent was then evaporated and theresidue was dried under high vacuum to afford the desired product,2-amino-4-[2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-1-(methoxycarbonylmethyl-carbamoyl)-ethylcarbamoyl]-butyricacid methyl ester, as a yellowish sticky solid. ¹H-NMR (D₃COD, 300 MHz)δ (ppm): 6.24 (d, J=10.5 Hz, 0.22H), 4.49-4.4.33 (m, 1H), 4.15-3.91 (m,3H), 3.76 (s, 3H), 3.65-3.57 (m, 2H), 3.51 (s, 3H), 3.21-2.73 (m, 2.8H),2.45-2.43 (m, 2H), 2.10-1.92 (m, 2H), 1.13-1.03 (m, 3H).

Example 18

[0651] 18A. Formula II where R¹ is COOH, R² is H, R³ isCH₂—S-keto-Pvruvate Decyl Ester, R⁴ is H, R⁵ is H,k is 0, m is 2,and nis 1

[0652] A mixture of 3-bromopyruvic acid (200 mg), decyl alcohol (300mg), and p-toluenesulfonic acid (20 mg) in benzene (80 mL) was heated toreflux for 8 h in the dark. After solvent removal, the residue waschromatographed to afford 320 mg of clear oil. NMR indicated the productcontained the desired compound and small amount of excess alcohol. Tothis obtained intermediate (320 mg) in methanol (degassed, 50 mL) wasadded an aqueous glutathione solution (250 mg in 5 mL of water). Theresulting cloudy solution was stirred at room temperature for 5 h. Thesolvent was then evaporated and the residue was chromatographed onsilica gel with methylene chloride/methanol (7:1 and then 1:1) to afford198 mg of the expected product,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-decyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid, as a white sticky solid.

[0653]¹H-NMR (300 MHz, DMSO-d₆) δ (ppm): 6.50 (s, 0.50H), 4.53-4.47 (m,1H), 4.12 (t, 2H), 3.77 (s, 2H), 3.75-3.65 (m, 2H), 3.19-2.83 (m, 2.5H),2.53 (s, 2H), 2.40-2.33 (m, 2H), 2.02-1.97 (m, 2H), 1.65-1.59 (m, 2H),1.26 (s, 14H), 0.87 (t, J=6 Hz, 3H). MS (ESI) m/z: (M+H⁺) 534.

[0654] 18B. Other Compounds of Formula Ia

[0655] Similarly, by following the procedures of Example 18A andsubstituting decyl alcohol with:

[0656] octadecyl alcohol,

[0657] 2-isopropyl-5-methyl-cyclohexanol

[0658] cyclopentanol,

[0659] pentanol,

[0660] butanol

[0661] isopropanol

[0662] hexanol

[0663] sec-butanol

[0664] 1-ethylpropanol, and

[0665] 10-(1,5-dimethyl-hexyl)-10a,11a-dimethyl-2,3,4,6,6a,7,7a,8,9,10,10a,11,11a,11b-tetradecahydro-1H-cyclopenta[b]phenanthren-3-ol there are obtainedthe following compounds, respectively:

[0666]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-octadecyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0667]2-amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(2-isopropyl-5-methylcyclohexyloxycarbonyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid, MS (ESI) m/z: 532 (M⁺+H, 100)

[0668]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-cyclopentyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid, MS (ESI) m/z: 662 (M⁺+H, 100)

[0669]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-oxo-2-pentyloxycarbonyl-ethylsulfanyl)-ethylcarbamoyl]-butyricacid, MS (ESI) m/z: 464 (M⁺+H, 100),

[0670]2-amino-4-[2-(2-butoxycarbonyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid, ¹H NMR (D₂O): 0.83-0.91 (m, 3H), 1.29-1.39 (m, 2H), 1.60-1.67 (m,2H), 2.12-2.18 (m, 2H), 4.47-2.60 (m, 2H), 2.80-3.0 (m, 1.5H), 3.04-3.20(m, 1H), 3.21-3.33 (m, 0.5H), 3.82-3.98 (m, 3H)4.14-4.28 (m, 2H),4.46-4.70 (m, 1H),

[0671]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-isopropoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid, MS (ESI) m/z: 436 (M⁺+H, 100),

[0672]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid, MS (ESI) m/z: 478 (M⁺+H, 100),

[0673]2-amino-4-[2-(2-sec-butoxycarbonyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid, MS (ESI) m/z: 450 (M⁺+H, 100),

[0674]2-amino-4-{1-(Carboxymethyl-carbamoyl)-2-[2-(1-ethyl-propoxycarbonyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid, and

[0675]2-amino-4-(1-(carboxymethyl-carbamoyl)-2-{2-[10-(1,5-dimethyl-hexyl)-10a,11a-dimethyl-2,3,4,6,6a,7,7a,8,9, 10,10a, 11,11 a,11b-tetradecahydro-1H-cyclopenta[b]phenanthren-3-yloxycarbonyl]-2-oxo-ethylsulfanyl}-ethylcarbamoyl)-butyricacid.

Example 19

[0676] 19A. Formula Ia where A is γ-Glu-CvsX-Gly, R is H, and X is aCovalent Bond; and Formula II where R¹ is COOH, R² is H, R³ isCH₂—S-Keto-pyruvate Ethyl Ester, R⁴ is H, R⁵ is H, k is 0 m is 2 , and nis 1.

[0677] Formula 203 where AA₁ is Gly

[0678] Following art-recognized procedures, NH-Fmoc-protected GlyOH (1eq) is dissolved in DCM and contacted with DCC (0.6 eq) in the presenceof a catalytic amount of DMAP to give the F-moc protected anyhdridecorresponding to Formula 202 where AA₁ is glycine, i.e.,O-(Gly-NHFmoc)₂. The anhydride so-prepared (10 eq) is dissolved in DCM,to which is added DIC (5 eq) in small portions, with stirring. Theresulting mixture is stirred for 1 h to give a clear solution that isthen added to Wang resin (1 eq, pre-swelled in DMF) in the presence ofDMAP (0.1 eq). The resulting resin suspension is shaken for 1 h, andthen thoroughly washed with DMF to afford the resin-bound,NH-Fmoc-protected glycine corresponding to Formula 203, which is carriedforward without further purification.

[0679] Formula 204 where AA₁ is Gly

[0680] The Fmoc protected, resin bound glycine of Formula 203 isde-protected using 20% piperidine in DMF followed by DMF wash (5 times)to give the corresponding resin-bound glycine of Formula 204, which iscarried forward without further purification.

[0681] Formula 206 where AA₁ is Gly, and AA₂ is Cys

[0682] The resin-bound glycine of Formula 204 (1 eq) is contacted withTBTU (2 eq), DIPEA (4 eq) and NH-Fmoc-protected, S-t-butylthio-cysteine(2 eq) (Formula 205), followed by DMF wash (3 times) to afford thecorresponding NH-Fmoc protected, resin bound di-peptide of Formula 206,which is carried forward without further purification.

[0683] Formula 207 where AA₁ is Gly, and AA₂ is Cys

[0684] The Fmoc protected, resin bound di-peptide of Formula 206 isde-protected using 20% piperidine in DMF followed by DMF wash (5 times)to give the corresponding resin-bound di-peptide of Formula 207, whichis carried forward without further purification.

[0685] Formula 209 where AA₁ is Gly, AA₂ is Cys, and AA₃ is γ-Glu

[0686] The resin-bound di-peptide of Formula 207 (1 eq) is contactedwith TBTU (2 eq), DIPEA (4 eq) and NH-Fmoc-protected glutamine (2 eq)(Formula 208), followed by DMF wash (3 times) to afford thecorresponding NH-Fmoc protected, resin bound tri-peptide of Formula 209,which is carried forward without further purification.

[0687] Formula 210 where AA₁ is Gly, AA₂ is Cys, and AA₃ is γ-Glu

[0688] The Fmoc protected, resin bound tri-peptide of Formula 209 isde-protected using 20% piperidine in DMF followed by DMF wash (5 times)to give the corresponding resin-bound tri-peptide of Formula 210, whichis carried forward without further purification.

[0689] Formula 301c where AA₁ is Gly, AA₂ is Cys, L is t-Butylthio, andAA₃ is γ-Glu

[0690] The resin-bound tri-peptide of Formula 210 (corresponding toFormula 300c where AA₂ is t-butylthio protected cysteine) is treatedwith 50% mercaptoethanol (in DMF) for 5 hours, followed by 10% DTT for 1hour to remove the t-butylthio protecting group, affording thecorresponding resin-bound tripeptide of Formula 301c, after filtrationand washed with DMF (3 times) and DCM (5 times).

[0691] Formula 302c where AA₁ is GlV, AA₂ is Cys, AA₃ is γ-Glu, and R isEthyl

[0692] To the resin-bound tripeptide of Formula 301 c, dissolved in DMF,is slowly added ethyl 3-bromopyruvate (Formula 102 where Halo is Bromoand R is Ethyl) (2 eq). The nucleophilic substitution is stopped byfiltration after 1 hour to afford the resin-bound tri-peptide conjugateof Formula 302c, which is washed with DMF (3 times), DCM (10 times), andMeOH (2 times), and dried under high vacuum for 10 hours.

[0693] Formula 303c where AA₁ is Gly, AA₂ is Cys, AA₃ is γ-Glu, and R isEthyl

[0694] The resin-bound tri-peptide conjugate of Formula 302c is cleavedfrom the resin by treatment for 3 hours using a cocktail containing 95%TFA (5% water). The solvent is partially removed and 50 fold of coldether is added to the mixture. The clear supernatant is removed and theprecipitate washed with cold ether (2 times). The resulting solid isdissolved in water, filtered through a pre-packed C18 short column, andlyophilized to afford the title compound of Formulae I and II, i.e.,2-amino-N-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethyl]-succinamicacid, as a fluffy white solid. ¹H-NMR (300 MHz, D₂O) δ (ppm): 4.63-4.47(m, 1H), 4.27-4.17 (m, 2H), 3.96 (s, 2H), 3.26-2.91 (m, 6H), 1.30-1.23(m, 3H). MS (ESI) m/z: 408 (M+H⁺).

[0695] 19B. Other Compounds of Formulae Ia and II

[0696] By following the procedures of Example 19A and substitutingFormulae 202, 205, 208, and/or 102 to introduce the desired,corresponding moieties at AA₁, AA₂, AA₃ and R, there are obtained thefollowing:

[0697]3-[2-[2-Amino-3-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-propionylamino]-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionicacid ethyl ester. ¹HNMR (300 MHz, D₂O) δ (ppm): 4.70-4.15 (m, 6H),3.96-3.92 (m, 4H), 3.19-2.86 (m 6H), 1.36-1.26 (m, 6H). MS (ESI) m/z:510 (M+H⁺).

[0698]3-[2-(2-Amino-3-mercapto-propionylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionicacid ethyl ester. ¹H-NMR (300 MHz, CD₃OD) δ (ppm): 4.69-4.55 (m, 1H),4.42-4.22 (m, 3H), 3.94 (s, 2H), 3.45-2.83 (m, 5H), 1.35-1.1.20 (m, 3H).MS (ESI) m/z: 396 (M+H⁺).

[0699]4-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid. ¹H-NMR (300 MHz, CD₃OD) δ (ppm): 4.68-4.38 (m, 1H), 4.23-4.19 (m,2H), 4.07-4.01 (m, 1H), 3.97-3.94 (m, 2H), 3.20-3.12 (m, 1H), 3.00-2.87(m, 2H), 2.53-2.59 (m, 2H), 2.21-2.10 (m, 2H), 1.28 (t, J=7.2 Hz, 3H).MS (ESI) m/z: 422 (M+H⁺).

[0700]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid ethyl ester. ¹H-NMR (300 MHz, D2O) δ (ppm): 6.80 (s, 0.13H),4.73-4.51 (m, 1H), 4.26-4.18 (m, 2H), 4.09-4.02 (m, 1H), 3.96 (s, 2H),3.29-83 (m, 2.5H), 2.56-2.49 (m, 2H), 2.21-2.12 (m, 2H), 1.24 (t, J=7.5Hz, 3H). MS (ESI) m/z: 422 (M+H⁺, 100).

[0701]4-[1-(Carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-4-(2-oxo-propionylamino)-buyricacid.

[0702]3-[2-(2-Amino-3-carboxy-propionylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacidethyl ester.

[0703]3-{2-Amino-2-[1-carboxy-2-(2-ethoxycarbonyl-2-hydroxy-vinylsulfanyl)-ethylcarbamoyl]-ethylsulfanyl}-2-hydroxy-acrylicacid ethyl ester.

[0704]2-Acetylamino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid. ¹H-NMR (D2O, 300 MHz) δ (ppm): 6.41 (s, 0.3H), 4.56-4.38 (m, 1H),4.20-4.05 (m, 3H), 3.83 (s, 2H), 3.19-2.72 (m, 3H), 2.27 (m, 2H),2.12-1.77 (m, 2H), 1.85 (s, 3H), 1.13 (m, 3H). MS(ESI) m/z: 465 (M+H⁺,100).

Example 20

[0705] 20A. Formula II where R¹ is COOH, R² is3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionyl, R³ isCH₂—S-Keto-pyruvate Ethyl Ester, R⁴ is H, R⁵ is H. k is 0, m is 2, and nis 1.

[0706] Formula 502c where AA₁ is Gly, AA₂ is Cys, and AA₃ is y-Glu. andR² is 3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionyl

[0707] A resin-bound tri-peptide of Formula 500c (made, e.g., asdescribed above with respect to Formula 210 in Example 19) is coupled,using pre-activated HOBt ester (DIC as dehydrating agent), with3-(6-hydroxy-2,7,8-trimethylchroman-2-yl)-propionic acid (a diphenolacid of Formula 501) to give the corresponding amino-substitutedresin-bound tri-peptide of Formula 502c.

[0708] Formula 503c where AA₁ is Gly, AA₂ is Cys, AA₃ is γ-Glu, and R²is 3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionyl

[0709] The resin-bound tri-peptide of Formula 502c is treated with 50%mercaptoethanol (in DMF) for 5 hours, followed by 10% DTT for 1 hour toremove the t-butylthio protecting group, affording the correspondingresin-bound tripeptide of Formula 503c, after filtration and washed withDMF (3 times) and DCM (5 times).

[0710] Formula 504c where AA₁ is Gly, AA₂ is CyS, AA₃ is γ-Glu, R² is3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionyl, and R is Ethyl

[0711] To the resin-bound tripeptide of Formula 503c, dissolved in DMF,is slowly added ethyl 3-bromopyruvate (Formula 102 where Halo is Bromoand R is Ethyl) (2 eq). The nucleophilic substitution is stopped byfiltration after 1 hour to afford the resin-bound tri-peptide conjugateof Formula 504c, which is washed with DMF (3 times), DCM (10 times), andMeOH (2 times), and dried under high vacuum for 10 hours.

[0712] Formula 505c where AA₁ is Gly, AA₂ is Cys, AA₃ is γ-Glu, R² is3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionyl and R is Ethyl

[0713] The resin-bound tri-peptide conjugate of Formula 504c is cleavedfrom the resin by treatment for 3 hours using a cocktail containing 95%TFA (5% water). The solvent is partially removed and 50 fold of coldether is added to the mixture. The clear supernatant is removed and theprecipitate washed with cold ether (2 times). The resulting solid isdissolved in water, filtered through a pre-packed C18 short column, andlyophilized to afford the expected compound of Formulae I and 11, i.e.,4-[1-(Carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-[3-(6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionylamino]-butyricacid. ¹H-NMR (300 MHz, CD₃OD) δ (ppm): 6.33 (s, 1H), 4.43-4.35 (m, 2H),4.20-4.14 (m, 2H), 3.93 (s, 2H), 3.35-3.05 (m, 2H), 2.98-2.83 (m, 2H),2.71-2.65 (m, 2H), 2.45-2.37 (m, 5H), 2.25-1.73 (m, 11H), 1.30-1.19 (m,6H). MS (ESI) m/z: 668 (M+H⁺).

[0714] 20B. Other Compounds of Formulae Ia and II

[0715] By following the procedures of Example 20A and substitutingFormulae 500, 501 and/or 102 to introduce the desired, correspondingmoieties at AA₁, AA₂, AA₃, R² and R, there is obtained the following:

[0716]4-[1-(Carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-{4-[2-(3,4-dihydroxy-phenyl)-vinyl]-benzoylamino}-butyricacid. ¹H-NMR (300 MHz, CD₃OD) δ (ppm): 7.71-7.69 (m, 2H), 7.20-7.09 (m,2H), 6.62-6.49 (m, 5H), 4.61-4.53 (m, 2H), 4.25-4.18 (m, 2H), 3.88 (d,J=4.2 Hz, 2H), 3.17-2.84 (m, 4H), 2.51-2.43 (m, 2H), 2.38-2.10 (m, 2H),1.33-1.16 (m, 3H). MS (ESI) m/z: 660 (M+H⁺).

Example 21

[0717] 21A. Formula II where R¹ is COOH, R² is4-[2-(3,4-dihydroxy-phenyl)-ethyl]-benzovl, R³ is CH₂—S-Keto-pyruvateEthyl Ester, R⁴ is H, R⁵ is H, k is 0, m is 2, and n is 1.

[0718] Formula 601 c where AA₁ is Gly, AA₉ is Cys, and AA₃ is γ-Glu, andR² is 4-[2-(3,4-dihydroxy-phenyl)-ethyl]-benzoyl

[0719] A resin-bound tripeptide of Formula 503c (prepared, for example,as described in Example 25A) is cleaved from the resin by treatment for3 hours using a TFA cocktail (TFA 93.5%, Tis 1.5%, EDT 2.5%, water2.5%). The solvent is partially removed and 50 fold of cold ether isadded to the mixture. The clear supernatant is removed and theprecipitate washed with cold ether (2 times). The resulting solid isdissolved in water, filtered through a pre-packed C18 short column, andlyophilized to afford the title compound of Formula 601 c, i.e.,4-[1-(carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-2-{4-[2-(3,4-dihydroxy-phenyl)-ethyl]-benzoylamino}-butyricacid. ¹H-NMR (300 MHz, CD₃OD) δ (ppm): 7.77-7.68 (m, 2H), 7.24-7.15 (m,2H), 6.85-6.43 (m, 3H), 4.62-4.56 (m, 2H), 3.95 (d, J=4 Hz, 2H),2.90-2.86 (m, 2H), 2.78-2.73 (m, 2H), 2.53-2.45 (m, 2H). MS (ESI) m/z:548 (M+H⁺).

[0720] Formula 602c where AA₁ is Gly, AA₂ is Cys, and AA₃ is γ-Glu, R²is 4-[2-(3,4-dihydroxy-phenyl)-ethyl]-benzoyl, and R is Ethyl

[0721] To the tripeptide of Formula 601c, dissolved in DMF, is slowlyadded ethyl 3-bromopyruvate (Formula 102 where Halo is Bromo and R isEthyl) (2 eq). The nucleophilic substitution is stopped by filtrationafter 1 hour to afford the resin-bound tri-peptide conjugate of Formula302c, which is washed with DMF (3 times), DCM (10 times), and MeOH (2times), and dried under high vacuum for 10 hours, to afford the titlecompound,4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-{4-[2-(3,4-dihydroxy-phenyl)-ethyl]-benzoylamino}-butyricacid.

[0722] 21 B. Other Compounds of Formula 601 c and Formulae Ia and II

[0723] By following the procedures of Example 21A and substitutingFormulae 500, 501 and/or 102 to introduce the desired, correspondingmoieties at AA₁, AA₂, M₃, R² and R, there are obtained the following:

[0724]4-[1-(Carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-2-[3-(6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionylamino]-butyricacid. ¹H-NMR (300 MHz, CD₃OD) δ (ppm): 6.34 (s, 1H), 4.54-4.3 (m, 2H),3.92 (d, J=3.9 Hz, 2H), 2.91-2.80 (m, 2H), 2.71-2.75 (m, 2H), 2.45-2.35(m, 4H), 2.25-1.68 (m, 9H), 1.23-1.16 (m, 6H). MS (ESI) m/z: 554 (M+H⁺).

[0725]4-[1-(Carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-[3-(6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionylamino]-butyricacid.

[0726]4-[1-(Carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-2-{4-[2-(3,4-dihydroxyphenyl)-vinyl]-benzoylamino}-butyricacid. ¹H-NMR (300 MHz, CD₃OD) δ (ppm): 7.40-7.08 (m, 4H), 6.79-6.46 (m,3H), 4.64-4.55 (m, 2H), 3.97 (s, 2H), 3.22-2.86 (m, 2H), 2.52-2.42 (m,2H), 2.40-2.02 (m, 2H). MS (ESI) m/z: 546 (M+H⁺).

[0727]4-[1-(Carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-2-{4-[2-(3,4-dihydroxy-phenyl)-vinyl]-benzoylamino}-butyricacid.

Example 22 Formula Ia where A is 5-Methyl-[1,3,4] thiadiazol-2-yl, R isEthyl, and X is S

[0728] A solution of 5-methyl-1,3,4-thiadiazole-2-thiol (264 mg, 2 mmol)and ethyl bromopyruvate (0.264 mL, 2.1 mmol) in methylene chloride (10mL) and acetonitrile (10 mL) was stirred at 20° C. for 3 hours undernitrogen. The precipitates were filtered, washed with methylenechloride, and dried under vacuum. This gave the expected product,3-(5-methyl-[1,3,4] thiadiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester (450 mg, yield 69%) as a solid. ¹H-NMR (300 Hz, D₃COD and d₆-DMSO)δ (ppm): 4.22 (q, J=7.1 Hz, 2H), 3.78 (d, J=14.1 Hz, 1H), 3.71 (d,J=14.1 Hz, 1H), 2.74 (s, 3H), 1.27 (t, J=7.1 Hz, 3H).

Example 23 Formula Ia where A is 5-Chloro-benzothiazol-2-yl, R is Ethyl,and X is S

[0729] A solution of 5-chloro-2-mercaptobenzothiozole (403 mg, 2 mmol)and ethyl bromopyruvate (0.264 mL, 2.1 mmol) in methylene chloride (10mL) and acetonitrile (30 mL) was shaken at 20° C. for 3 hours undernitrogen. The obtained solid was filtered, washed with methylenechloride, dried under vacuum. This gave the expected product,3-(5-chloro-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethyl ester(425 mg, yield 54%) as a solid. ¹H-NMR (300 Hz, D₃COD and d₆-DMSO) δ(ppm): 7.92 (d, J=8.6 Hz, 1H), 7.90 (d, J=2.1 Hz, 1H), 7.40 (dd, J=2.1Hz, J=8.6 Hz, 1H), 4.20 (d, q, J=2.1 Hz, J=7.1 Hz, 2H), 3.96 (d, J=13.1Hz, 1H), 3.86 (d, J=13.1 Hz, 1H), 1.23 (t, J=7.1 Hz, 3H).

Example 24 Formula Ia where A is 4,5-Dihydro-thiazol-2-yl, R is Ethyl,and X is S

[0730] A solution of 2-mercaptothiazole (238 mg, 2 mmol) and ethylbromopyruvate (0.264 mL, 2.1 mmol) in methylene chloride (7 mL) wasshaken at 20° C. for 3 hours under nitrogen. The obtained solid wasfiltered, washed with methylene chloride, and dried under vacuum. Thisgave the expected product,3-(4,5-dihydro-thiazol-2-ylsulfanyl)-2-oxo-propionic acid ethyl ester(440 mg, yield 71%) as a solid. ¹H-NMR (300 Hz, D₃COD) δ (ppm): 4.63 (d,J=13.1 Hz, 1H), 4.30-4.15 (m, 6H), 4.10 (d, J=13.1 Hz, 1H), 1.34 (t,J=7.1 Hz, 3H). MS (ESI) m/z: 234 (M+H, 100).

Example 25 Preparation of2-hydroxy-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-2,3-dihydro-furan-2,5-dicarboxylicacid diethyl ester

[0731] A solution of 2-mercapto-1-methylimidazole (228 mg, 2 mmol),ethyl bromopyruvate (0.266 mL, 2.1 mmol), and imidazole (150 mg, 2.2mmol) in methylene chloride (6 mL) and acetone (2 mL) was shaken at 20°C. under nitrogen for 4 hour. The solvents were evaporated under vacuum.The residue was treated with ethyl acetate and the liquid layer wasdiscarded. The residue was then dissolved in methylene chloride. Thesolution was washed with aqueous sodium bicarbonate solution and waterbefore dried over magnesium sulfate. Evaporation and chromatography(silica gel, methylene chloride-methanol 100:3 to 100:10 as the eluents)gave 20 mg of product. ¹HNMR (300 Hz, Cl₃CD) δ (ppm): 8.13 (br. s, 1H),7.01 (d, J=1.3 Hz, 1H), 6.91 (d, J=1.3 Hz, 1H), 4.65 (d, J=10.4 Hz, 1H),4.57 (d, J=10.4 Hz, 1H), 4.35 (q, J=7.1 Hz, 2H), 4.09 (q, J=7.1 Hz, 2H),3.69 (s, 3H), 1.38 (t, J=7.1 Hz, 3H), 1.21 (t, J=7.1 Hz, 3H). ¹³CNMR (75Hz, Cl₃CD) δ (ppm): 171.2, 159.6, 147.5, 138.9, 128.8, 122.8, 117.2,85.8, 80.3, 62.2, 62.1, 34.0, 14.2,14.1. MS (ESI) m/z 343 (M+1, 100),365 (M+Na, 7).

Example 26 Preparation of2,2-Dimethyl-3,4-dihydro-2H-[1,4]thiazine-3,5-dicarboxylic acid

[0732] To a solution of L-penicillamine (298 mg, 2.0 mmol) in water (5mL) and acetonitrile (5 mL) was added ethyl 3-bromopyruvate (0.25 mL,390 mg, 2.0 mmol) slowly. To the resulted mixture was added sodiumbicarbonate aqueous solution till the pH about 5. Acetonitrile wasremoved under vacuum and some more water was added. The solution wasextracted with methylene chloride. The water phase was separated andfreeze-dried under high vacuum. The residue was chromatographed (silicagel, methylene chloride-methanol-acetic acid 100:5:0.3 to 100:10:0.3) togive 150 mg of product (yield 31%). ¹H-NMR (300 Hz, D₃COD) δ (ppm) 6.17(s, 1H), 4.23 (q, J=7.1 Hz, 2H), 3.79 (s, 1H), 1.48 (s, 3H), 1.31 (t,J=7.1 Hz, 3H), 1.29 (s, 3H). ¹³CNMR (75 Hz, D₃COD) δ (ppm) 173.8, 164.0,128.0, 102.4, 64.1, 62.2, 41.2, 28.3, 25.2, 14.7 ppm. MS (ESI) m/z 268(M+Na, 100), 246 (M+1, 5).

Example 27 Preparation of4-[2-[2-(Adamantan-1-ylmethoxycarbonyl)-2-oxo-ethylsulfanyl]-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-2-amino-butyricacid

[0733] A solution of 10 mmol of 1-admantan-1-yl-methanol, 5 mmol ofbromo pyruvic acid, 20 mg of TsOH in 80 mL of benzene was heated toreflux under azeotropic condition for 6 to 12 h under nitrogenatmosphere. After cooling, the solvent was removed under reducedpressure. The residue was chromatographed to afford the bromo pyruvateester intermediate.

[0734] The above prepared pyruvate ester intermediate(1 mmol) wasdissolved in 5-10 mL of acetonitrile. This solution was then slowlyadded to a solution of 1 mmol of glutathione in 10 mL of deionized waterunder vigorous stirring. Upon the completion of the addition, theresulting mixture was stirred for 3-8 h. The reaction was quenched byadding 50 mL water and washed with ether (3×20 mL). The aqueous layerwas filtered through a C18 pad and freeze-dried. The crude product waschromatographed using reverse phase column using a manual gradient from100% water to 70/30 water/acetonitrile to afforded a white sticky solid.MS (ESI) m/z 542 (M+H, 100), 560 (M+H₂O, 40).

Example 28 Preparation of1-[3-(2-Ethoxycarbonyl-2-oxo-ethylsulfanyl)-2-methyl-propionyl]-pyrrolidine-2-carboxylicacid

[0735] This compound is prepared using similar procedure described inExample 4.

[0736]¹H-NMR (300 Hz, D₃COD) δ (ppm) 6.37 (s, 0.35H), 4.43 (dd, 1H),4.21 (q, 2H), 3.73 (m, 3H), 2.75-3.16 (m, 3H), 2.21 (m, 1H), 2.03 (m,3H), 1.31 (t, 3H), 1.16 (dd, 3H). MS (ESI) m/z 332 (M+H, 100).

Example 29

[0737] 29A. Preparation of2-Amino-4-[1-carboxy-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0738] To a solution of degassed and nitrogen purged1-carboxy-3-(1-carboxy-2-mercapto-ethylcarbamoyl)-propyl-ammonium;trifluoro-acetate (50 mg, 85%, 0.12 mmol) in 1:1 acetonitrile and water(2 mL) was added of ethyl bromo pyruvate (14.6 μL). The reaction wasallowed to stir for 1 hour before it was stopped and extracted withether. The water phase was then directly filtered through a short solidphase column and placed on the lyopholyzer overnight to yield whitecrystals (55 mg, 95% yield). ¹HNMR (300 Hz, D₃COD) δ (ppm) 6.37 (s,0.15H), 4.62 (m, 1H), 4.23 (q, 2H), 3.92 (m, 1H), 2.83-3.30 (m, 2.4H),2.56 (m, 2H), 2.17 (m, 2H), 1.31 (t, 3H).

[0739] 29B. Other Compounds of Formulae Ia and II

[0740] By following the procedures of Example 29A and substituting1-carboxy-3-(1-carboxy-2-mercapto-ethylcarbamoyl)-propyl-ammonium;trifluoro-acetate with (2-amino-3-mercapto-propionylamino)-acetic acidand2-amino-4-[1-(ethoxycarbonylmethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-butyricacid, respectively, there are obtained the following:

[0741]3-[2-Amino-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionicacid ethyl ester White crystals (97% yield). ¹HNMR (300 MHz, D₃COD) δ(ppm): 6.42 (s, 0.36H), 4.61 (m, 1H), 4.14-4.27 (m, 4H), 4.01 (m, 1H),3.95 (s, 2H), 2.80-3.20 (m, 2.56H), 2.60 (m, 2H), 2.20 (m, 2H), 1.26(tt, 6H).

[0742]2-Amino-4-[1-(ethoxycarbonylmethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid White solid (41% yield). ¹HNMR (300 MHz, D₃COD) δ (ppm): 6.42 (s,0.36H), 4.38 (dd, 1H), 4.28 (q, 2H), 2.95-3.54 (m, 2H), 1.27 (t, 3H). MS(ESI) m/z 293 (M+H, 100).

Example 302-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0743] 30A. Formula Ib where A is -Glu-CysX-Gly, W═NOR^(a) where R^(a)is H, and Z═OR where R is Ethyl

[0744]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid (1.0 mmol) was dissolved in 10 mL water, to which was added, atambient temperature, a solution of (1.2 mmol,1.2eq) hydroxylaminehydrochloride. The mixture was agitated for 24 hrs. LC/MS of thereaction aliquot indicated the completion of the reaction. The mixturewas then loaded onto a Cl 8 reversed-phase column and eluted withacetonitrile and water, each containing 0.2% TFA (0 to 10 min, 5%acetonitrile; 10 to 50 min, 60% acetonitrile; 60 to 70 min, 100%acetonitrile). The fractions containing pure compound were pooled,vacuum distilled at 30° C. to one fourth of the volume and lyophilizedto obtain the corresponding oxime as its TFA salt.

[0745] The oxime TFA salt (1.0 mmol) in 20 mL of water was treated with1.2 mmols of dilute (1.0 M) HCl at 0° C. and the resulting clearsolution was freeze-dried to obtain the desired title compound,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid, as a white solid. ¹H NMR (D₂O): δ 1.28 (t, J=6.0 Hz, 3H),2.10-2.27 (m, 2H), 2.50-2.59 (m, 2H), 2.82-3.06 (m, 2H), 3.64 (q, J=15.0Hz, 2H), 3.97 (s, 2H), 4.06 (t, J=6.0 Hz, 1H), 4.29 (q, J=6.0 Hz),4.58-4.64 (m, 1H).MS(ESI) m/z: 437 (M+H, 100%).

[0746] 30B. Formula Ib where A is γ-Glu-CvsX-Glv, W═NOR^(a) where R^(a)is H, and Z═OR where R is n-Butyl

[0747] Similarly, by following the procedure of Example 30A andsubstituting2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid with2-amino-4-[2-(2-butoxycarbonyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid there is obtained the following compound: 2-Amino-4-[2-(2-butoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid; compound with HCl; ¹H NMR (D₂O): δ 0.60-0.75 (m, 3H), 1.05-1.22(m, 2H), 1.40-1.55 (m, 2H), 1.92-2.15 (m, 2H), 2.35-2.55 (m, 2H),2.26-2.27 (m, 1H), 2.80-2.92 (m, 1H), 3.43 (q, J=12 Hz, 2H), 3.80 (s,2H), 3.85-3.99 (m, 1H), 4.01-4.15 (m, 2H), 4.40-4.55 (m, 1H). MS(ESI)m/z: 465 (M+H, 100%), 466 (M+2H, 25%).

[0748] 30B. Formula Ib Varying R^(a)

[0749] Similarly, by following the procedure of Example 30A andsubstituting hydroxylamine HCl with another substituted hydroxylaminethere are obtained the following compounds:

[0750]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid; ¹H NMR (D₂O): δ 1.28 (t, J=9.0 Hz, 3H), 2.10-2.23 (m, 2H),2.51-2.59 (m, 2H), 2.70-2.89 (m, 1H), 2.95-3.05 (m, 1H), 3.57-3.64 (m,2H), 3.84 (s, 1H), 3.88-4.16 (m, 6H), 4.32 (q, J=9.0 Hz, 2H), 4.54-4.0(m, 1H).

[0751]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-phenoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid; compound with HCl; ¹H NMR (D₂O): δ 1.15 (t, J=6.9 Hz, 3H),2.0-2.14 (m, 2H), 2.21-2.50 (m, 2H), 2.65-2.80 (m, 2H), 2.81-2.98 (m,2H), 3.6 (brs, 2H), 3.65-3.99 (m, 3H), 4.10-4.21 (m, 2H), 4.45-4.60 (m,1H), 6.84-7.30 (m, 5H). MS(ESI) m/z: 513 (M+H, 100%), 514 (M+2H, 30%).

[0752]2-Amino-4-[2-(2-benzyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid; compound with HCl; ¹H NMR (D₂O): δ 1.05 (t, J=9.0 Hz, 3H),1.99-2.20 (m, 2H), 2.31-2.50 (m, 2H), 2.52-2.71 (m, 2H), 2.73-2.97 (m,2H), 3.20-3.48 (m, 2H), 3.60-4.15 (m, 5H), 4.31-4.50 (m, 1H), 5.03 (s,2H), 6.95-7.20 (m, 5H). MS(ESI) m/z: 527 (M+H, 100%), 528 (M+2H, 30%).

[0753]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-ethoxycarbonyl-2-(4-nitrobenzyloxyimino)-ethylsulfanyl]-ethylcarbamoyl}-butyricacid; ¹H NMR (D₂O): δ 1.08 (t, J=6 Hz, 3H), 2.0-2.20 (m, 2H), 2.35-2.60(m, 2H), 2.62-2.76 (m, 1H), 2.80-3.10 (m, 1H), 3.40-3.61 (m, 2H),3.70-4.21 (m, 5H), 4.50 (brs, 1H), 5.16 (s, 2H), 7.27 (d, J=9.0 Hz, 2H),7.80 (d, J=9.0 Hz, 2H). MS(ESI) m/z: δ 572 (M+H, 100%), 573 (M+2H, 30%).

[0754]4-[2-(2-Allyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-2-amino-butyricacid; compound with di-HCl; ¹H NMR (D₂O): δ 1.20 (t, J=7.2 Hz, 3H),2.08-2.20 (m, 2H), 2.45-2.60 (m, 2H), 2.65-2.80 (m, 1H), 2.90-3.06 (m,1H), 3.50-3.64 (m, 2H), 3.90 (s, 2H), 4.10 (t, J=6.9 Hz, 1H), 4.22 (q,J=7.2 Hz, 2H), 4.49-4.54 (m, 1H), 4.70 (d, J=7.0 Hz, 2H), 5.15-5.30 (m,2H), 5.80-6.10 (m, 1H). MS(ESI) m/z: 477 (M+H, 100%), 478 (M+2H, 30%).

[0755]2-Amino-4-[2-(2-tert-butoxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid; compound with di-HCl; ¹H NMR (D₂O): δ 1.10-1.20 (m, 12H),2.03-2.25 (m, 2H), 2.40-2.56 (m, 2H), 2.63-2.75 (m, 1H), 2.90-3.16 (m,1H), 3.56-3.78 (m, 2H), 3.88 (s, 2H), 4.20 (t, J=6.9 Hz, 1H), 4.30 (q,J=7.2 Hz, 2H), 4.40-4.56 (m, 1H). MS(ESI) m/z: 493 (M+H, 100%), 494(M+2H, 30%).

[0756]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-ethoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid; compound with di-HCl; ¹H NMR (D₂O): δ 1.15 (t, J=7.2 Hz, 3H), 1.19(t, J=7.2 Hz, 3H), 1.10-1.30 (m, 2H), 1.50-1.62 (m, 2H), 1.72-1.87 (m,1H), 2.0-2.10 (m, 1H), 2.61 (d, J=6 Hz, 2H), 2.96 (s, 2H), 3.10 (t,J=9.0, 1H), 3.21-3.34 (m, 4H), 3.55-3.26 (m, 1H). MS(ESI) m/z: 465 (M+H,100%), 466 (M+2H, 30%).

[0757] 30C. Formula Ib Varying R and R^(a)

[0758] Similarly, by following the procedure of Example 30A andsubstituting2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid with2-amino-4-[2-(2-butoxycarbonyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid and hydroxylamine HCl with another substituted hydroxylamine thereare obtained the following compounds:

[0759]2-Amino-4-[2-(2-butoxycarbonyl-2-methoxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid; ¹H NMR (DMSO-d₆): δ 0.90 (t, J=6.0 Hz, 3H), 1.25-1.42 (m, 2H),1.5-1.72 (m, 2H), 1.95-2.14 (m, 2H), 2.20-2.80 (m, 4H), 2.82-3.03 (m,1H), 3.4-3.69 (m, 3H), 3.7-3.82 (m, 2H), 3.85-4.0 (m, 4H), 4.12-4.25 (m,2H), 4.46-4.65 (m, 1H). MS(ESI) m/z: 479 (M+H, 100%), 480 (M+2H, 30%)

[0760]2-Amino-4-[2-(2-benzyloxyimino-2-butoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid; ¹H NMR (DMSO-d₆): δ 0.90 (t, J=6.0 Hz, 3H), 1.30-1.45 (m, 2H),1.52-1.70 (m, 2H), 2.10 (brs, 2H), 2.30-2.55 (m, 2H), 2.66-2.81 (m, 1H),2.90-3.05 (m, 1H), 3.50-3.75 (m, 2H), 3.77-3.81 (m, 2H), 3.87 (brs, 1H),4.10-4.23 (m, 2H), 4.45-4.68 (m, 1H), 5.28 (s, 3H), 7.30 (brs, 5H).MS(ESI) m/z: 555 (M+H, 100%), 556 (M+2H, 30%)

Example 31 Alternative Synthesis of2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0761] A mixture of bromopyruvic acid ethyl ester (390 mg) andhydroxyamine hydrochloride (1.05 eq) was stirred for 3 h at rt. Thewhite solid was filtered and washed with water and dried (420 mg). Toglutathione (307 mg) in degassed water (10 mL) was added above madeoxime, 3-bromo-2-hydroxyimino-propionic acid ethyl ester (1 eq) in smallportions. After 3 h, the reaction was washed with EtOAc (10 mL) andfreeze-dried to afford2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid as a fluffy white solid (179-6, 410 mg). ¹H NMR (D₂O): δ 1.28 (t,J=6.0 Hz, 3H), 2.10-2.27 (m, 2H), 2.50-2.59 (m, 2H), 2.82-3.06 (m, 2H),3.64 (q, J=15.0 Hz, 2H), 3.97 (s, 2H), 4.06 (t, J=6.0 Hz, 1H), 4.29 (q,J=6.0 Hz), 4.58-4.64 (m, 1H).

Example 321-{3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionyl}-pyrrolidine-2-carboxylicacid methyl ester

[0762] 32A. Formula Ic where A is γ-Glu-CysX-Glv, W is ═O, and Z isNR^(b)R^(c) Forming a 5-Membered Acyl-substituted Ring

[0763] To a solution of proline HCl salt (452 mg, 3.5 mmol) in a mixedsolvent of 3 mL of acetonitrile and 5 mL of DCM in an ice bath was addeddropwise a chilled triethylamine solution (353 mg, 3.5 mmol) withstirring. Upon completion of the triethylamine, the resulting suspensionwas allow to stir for 5 min and stored in the ice bath.

[0764] To 740 mg (4 mmol) of bromopyruvic acid dissolved in 5 mL ofacetonitrile in an ice bath was added slowly the above prepared aminoacid. Upon the completion of the amino acid addition, EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride)(806 mg,4.2 mmol) was added to the mixture in small portions with vigorousstirring. The ice bath was removed and the mixture was allowed to stirfor 20 min at rt. The reaction was quenched by adding 40 mL of water andthe mixture was extracted with ethylacetate (3×30 mL). The organiclayers were dried over Na₂SO4 followed by solvent removal under reducedpressure. The residue was chromatographed using hexanes/ethylacetate(2:1) to afford a thick yellow oil (473 mg, 46%).

[0765] A solution of glutathione(2-amino-4[1-(carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-butyricacid)(368 mg, 1.2 mmol) in 5 mL of water was degassed through highvacuuming and argon purging. To this solution, the above prepared amidein 1.5 mL acetonitrile was added with vigorous stirring. The reactionwas monitored with periodic MS checking until the glutathione wascompletely consumed (3 h). The reaction was quenched by adding 40 mL ofwater and the resulting mixture was extracted with ethylacetate (2×30mL) and hexanes (2×20 mL). The aqueous solution was then filteredthrough a cotton pad and freeze-dried to afford1-{3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionyl}-pyrrolidine-2-carboxylicacid methyl ester; compound with HBr;as a yellow sticky solid (642 mg,91%). ¹H-NMR (D2O, 300 MHz) δ (ppm): 4.85-4.70 (m, 0.5H), 4.47-4.38 (m,1.5H), 3.93 (t, J=6.5 Hz, 1H), 3.85 (s, 2H), 3.66-3.43 (m, 5.7H),2.91-2.63 (m, 2H), 2.44 (m, 2H), 2.20-1.80 (m, 6H; MS(ESI) m/z: 505(M+H⁺, 100).

[0766] 32B. Formula Ic Varving NR^(b)R^(c)

[0767] Similarly, by following the procedure of Example 32A andsubstituting proline with another heterocyclic amine there is obtainedthe following compounds:

[0768]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[3-(4-methyl-piperidin-1-yl)-2,3-dioxo-propylsulfanyl]-ethylcarbamoyl}-butyricacid. ¹H-NMR (D2O, 300 MHz) δ (ppm): 4.51 (m, 1H), 4.17-4.13 (m, 1H),3.95-3.91 (m, 3H), 3.71-3.60 (m, 1.6H), 3.55-3.42 (m, 1H), 3.09 (m, 1H),2.98-2.90 (m, 1H), 2.85-2.72 (m, 2H), 2.48 (m, 2H), 2.13 (m, 2H),1.70-1.57 (m, 3H), 1.06 (m, 2H), 0.85-0.83 (m, 3H). MS(ESI) m/z: 475(M+H+, 100).

[0769]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-pyrrolidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid; ¹H-NMR (D2O, 300 MHz) δ (ppm): 4.56 (dd, J=8.2, 5.1 Hz, 1H), 4.04(t, J=6.6 Hz, 1H), 3.98 (s, 2H), 3.79 (s, 1H), 3.61 (t, J=5.6 Hz, 2H),3.46 (t, J=5.6 Hz, 2H), 3.00 (dd, J=14.0, 5.1 Hz, 1H), 2.87 (dd, J=14.0,8.7 Hz, 1H), 2.56 (m, 2H), 2.19 (m, 2H), 1.89 (m, 4H). MS(ESI) m/z: 447(M+H⁺, 100).

[0770]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(3-morpholin-4-yl-2,3-dioxo-propylsulfanyl)-ethylcarbamoyl]-butyricacid; MS(ESI) m/z: 463 (M+H⁺, 100).

[0771]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid; ¹H-NMR (D2O, 300 MHz) δ (ppm): 4.49 (dd, J=8.3, 5.0 Hz, 1H),3.69-3.61 (m, 3.5H), 3.41 (t, J=5.7 Hz, 2H), 3.28 (t, J=5.5 Hz, 2H),2.92 (dd, J=14.0, 5.0 Hz, 1H), 2.77 (dd, J=14.0, 8.6 Hz, 1H), 2.39 (m,2H), 2.01 (q, J=7.3 Hz, 2H), 1.57-1.43 (m, 6H). MS(ESI) m/z: 461 (M+H⁺,100).

Example 332-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-dimethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0772] 33A. Formula Ic where A is γ-Glu-CvsX-Glv, W is ═O, and Z isNR^(b)R^(c) where R^(b) and R^(c) are Methyl

[0773] To 185 mg (1 mmol) of bromopyruvic acid dissolved in 2 mL of DMFcooled in a ice bath, was added slowly a solution of N,N-dimethylaminein DCM (43 mg in 2 mL). The ice bath was removed and the mixture wasallowed to warm to room temperature. To this mixture was simultaneouslyadded Bop-Cl (Bis(2-oxo-3-oxasolidinyl)phosphonic chloride) (279 mg, 1.1mmol) and triethylamine (106 mg, 1.05 mmol) with triethylamine additionslightly ahead over a period of 15 min. Upon the completion of thereagent addition, the reaction was allowed to stir for another 25 minbefore being quenched by addition of 40 mL water and 40 ethylacetate.After stirring for 2 min, the layers were separated, the organic layerwas washed with water (2×20 mL), and the combined aqueous layers wereback-extracted with ethylacetate (40 mL). The combined organic layerswere dried over Na₂SO4 and concentrated. The crude product waschromatographed with EtOAc/Hexanes to afford a pale yellow oil (88 mg,48%).

[0774] A solution of glutathione (170 mg, 0.55 mmol) in 2 mL of waterwas degassed through high vacuuming and argon purging. To this solution,amide (128 mg, 0.66 mmol, prepared as above) in 1 mL acetonitrile wasadded with vigorous stirring. The reaction was monitored with periodicMS checking until the GSH was completely consumed (1.5). The reactionwas quenched by adding 30 mL of water and the resulting mixture wasextracted with ethylacetate (2×20 mL) and hexanes (2×15 mL). The aqueoussolution was then filtered through a cotton pad and freeze-dried toafford a pale yellow sticky solid of2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-dimethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyric acidhydrobromide salt (262 mg, 95% yield, 179-91). The above product waspurified by RP-LC and converted to HCl salt using a 0.1 M HCl solution.The purified product was obtained as a pale yellow sticky solid. ¹H-NMR(D2O, 300 MHz) δ (ppm): 4.45 (dd, J=8.3, 5.2 Hz, 1H), 3.94 (t, J=6.6 Hz,1H), 3.86 (s, 2H), 3.66-3.64 (m, 1.6H), 2.92-2.71 (m, 2H), 2.90 (s, 3H),2.84 (s, 3H), 2.53-2.36 (m, 2H), 2.18-1.99 (m, 2H).), MS(ESI) m/z: 421(M+H+, 100).

[0775] 33B. Formula Ic Varying NR^(b)R^(c)

[0776] Similarly, by following the procedure of Example 33A andsubstituting of N,N-dimethylamine with another amine there are obtainedthe following compounds:

[0777]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-diethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid ¹H-NMR (D2O, 300 MHz) δ (ppm): 4.45 (dd, J=8.4, 5.2 Hz, 1H), 3.92(t, J=6.5 Hz, 1H), 3.85 (s, 2H), 3.66-3.64 (m, 1.7H), 3.27 (q, J=7.2 Hz,2H), 3.19 (q, J=7.1 Hz, 2H), 2.92-2.71 (m, 2H), 2.44 (m, 2H), 2.08 (m,2H), 1.07-0.97 (m, 6H). MS(ESI) m/z: 449 (M+H+, 100).

[0778]2-Amino-4-(1-(carboxymethyl-carbamoyl)-2-{2-[2-(4-hydroxy-phenyl)-1-methoxycarbonyl-ethylcarbamoyl]-2-oxo-ethylsulfanyl}-ethylcarbamoyl)-butyricacid; ¹H--NMR (D2O, 300 MHz) δ (ppm):7.13-7.07 (m, 5H), 6.81-6.76 (m,2H), 6.29 (s, 0.1H), 4.67 (dd, J=8.8, 5.4 Hz, 1), 4.52-4.46 (m, 1H),4.03 (t, J=6.3 Hz, 1H), 3.97 (s, 2H), 3.72-3.66 (m, 3.4H), 3.17 (dd,J=14.4, 5.0 Hz, 1H), 3.00-2.90 (m, 2H), 2.78-2.66 (m, 2H), 2.56-2.49 (m,2H), 2.18 (m, 2H); MS(ESI) m/z: 571 (M+H+, 100), 589 (M+H₂O+H⁺, 45), 601(M+MeOH+H⁺, 32).

[0779]2-{3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionylamino}-3-methyl-pentanoicacid methyl ester ¹H-NMR (D2O, 300 MHz) δ (ppm): 4.56 (dd, J=8.5, 5.1Hz, 1H), 4.28-4.20 (m, 1H), 3.85-3.82 (m, 3H), 3.66-3.61 (m, 3.4H),2.99-2.71 (m, 3.1H), 2.46-2.39 (m, 2H), 2.09-2.03 (m, 2H), 1.90-1.84 (m,1H), 1.35-1.25 (m, 1H), 1.15-1.02 (m, 1H), 0.79-0.71 (m, 6H). MS(ESI)m/z: 535 (M+H⁺, 100), (M+H₂O+H⁺, 20), (M+MeOH+H⁺, 40).

[0780]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-octylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid; ¹H-NMR (D2O, 300 MHz) δ (ppm): 6.20 (s, 0.02H), 4.47-4.39 (m, 1H),3.78-3.64 (m, 3H), 3.38 (m, 0.8H), 3.15-3.05 (m, 2H), 2.90-2.82 (m, 1H),2.65-2.55 (m, 1H), 2.49 (m, 1H), 2.32-2.25 (m, 2H), 1.95-1.84 (m, 2H),1.46-1.37 (m, 2H), 1.28-1.15 (m, 10H), 0.83 (t, J=13 Hz, 3H). MS(ESI)m/z: 505 (M+H⁺, 100), 523 (M+H₂O+H⁺, 45), 537 (M+MeOH+H⁺, 38).

[0781]2-Amino-4-1-(carboxymethyl-carbamoyl)-2-(2-ethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid; MS(ESI) m/z: 421 (M+H⁺, 100)

[0782]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(1-methoxycarbonyl-2-phenyl-ethylcarbamoyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid; ¹H-NMR (D2O, 300 MHz) δ (ppm): 7.23-7.09 (m, 5H), 6.15 (s, 0.05H),4.63-4.58 (m, 1H), 4.48-4.33 (m, 1H), 3.93-3.89 (m, 1H), 3.86-3.80 (m,2H), 3.61-3.43 (m, 3H), 3.21-3.12 (m, 1H), 2.97-2.87 (m, 1H), 2.84-2.55(m, 3H), 2.44-2.36 (m, 2H), 2.1-2.02 (m, 2H); MS(ESI) m/z: 555 (M+H+,100), 573 (M+H₂O+H⁺, 36), 587 (M+MeOH+H⁺, 85).

[0783]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-cyclohexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid; ¹H-NMR (D2O, 300 MHz) δ (ppm): 6.18 (s, 0.05H), 4.44-4.39 (m, 1H),3.94 (t, J=6.5 Hz, 1H), 3.85 (s, 2H), 3.65 (s, 0.6H), 3.54-3.41 (m, 1H),2.97-2.71 (m, 3H), 2.47-2.41 (m, 2H), 2.12-2.03 (m, 2H), 1.66-1.1.42 (m,5H), 1.22-0.97 (m, 5H); MS(ESI) m/z: 475 (M+H⁺, 100), 493 (M+H₂O+H⁺,52), 507 (M+MeOH+H⁺, 15).

[0784]2-Amino-4-[2-(2-benzylcarbamoyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid; ¹H-NMR (D2O, 300 MHz) δ (ppm):7.26-7.16 (m, 5H), 4.69-4.36 (m,1H), 4.30-4.27 (m, 2H), 3.91 (t, J=13.2 Hz, 1H), 3.81 (s, 2H), 3.64 (s,0.5H), 2.86-2.80 (m, 2H), 2.70-2.64 (m, 1H), 2.06 (m, 2H); MS(ESI) m/z:483 (M+H⁺, 100), 501 (M+H₂O+H⁺, 64), 515 (M+MeOH+H⁺, 52).

[0785]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid; ¹H-NMR (D2O, 300 MHz) δ (ppm): 6.19 (s, 0.02H), 4.47-4.40 (m, 1H),3.90-3.85 (m, 3H), 3.65 (d, J=2.5 Hz, 0.6H), 3.15-3.05 (m, 2H),2.97-2.71 (m, 3H), 2.46-2.40 (m, 2H), 2.11-2.01 (m, 2H), 1.39-1.32 (m,2H), 1.18-1.08 (m, 6H), 0.72-0.68 (m, 3H). MS(ESI) m/z: 477 (M+H⁺, 100),495 (M+H₂O+H⁺, 68), 509 (M+MeOH+H⁺, 70).

Example 342-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hydroxyimino-3-oxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid

[0786] To a cooled (0° C.) solution of 3-bromopyruvic acid (0.835 g,0.005 mol) in acetonitrile (8 mL) was added a pre-cooled (0° C.)solution of piperidine (0.341 g, 0.004 mol) in CH₂Cl₂ (3 mL) slowlydrop-wise during 5 min. EDC was then added pinch-wise during 5 min. andthe mixture was stirred at the same temperature for 10 min. and themixture slowly warmed to eoom temperature. Quenching of the reactionwith 10 mL of cold water, extraction with ethylacetate (2×25 mL)followed by concentration and flash chromatography over silica gelafforded the desired product, 3-bromo-1-piperidin-1-yl-propane-1,2-dioneas a brown oil. The product was carried forward to the next step withoutassessing yield and purity.

[0787] To a cooled (0° C.) solution of glutathione (500 mg, 1.595 mmol)in water (15 mL) was added bromopyruvic acid amide (obtained from theabove reaction) in acetonitrile (10 mL) slowly drop-wise during 5 min.and the mixture stirred at the same temperature for 1 hr and slowlywarmed to room temperature and continued stirring overnight. LCIMSindicated the formation of the desired product,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid. The mixture was purified by MPLC to obtain a pale yellow solid asits TFA salt 400 mg, (43.7%).

[0788]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid as its TFA salt (400 mg, 0.696 mol) was dissolved in 8 mL of waterto which was added 1.3 eq. of solid NH₂OH.HCl at 0° C. and thehomogeneous solution was left standing over-night. LC/MS indicated thatthe reaction was incomplete. An additional 1 eq. of NH₂OH.HCl was addedat room temperature and the homogeneous solution was left standingovernight. LC/MS indicated complete conversion of the starting amide tothe corresponding oxime. The aqueous mixture was then passed throughreversed-phase C18 column using water and acetonitrile mixture asgradient eluent. The pure fractions were pooled, concentrated undervacuum at room temperature to one fourth of the volume and finallyfreeze-dried to obtain2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hydroxyimino-3-oxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid as its TFA salt (390 mg, 95.1%).

[0789] To a cooled (0° C.) solution of the above TFA salt (390 mg, 0.662mmol) in 10 mL of water was added a pre-cooled aqueous 1 N HCl (0.662mol) and the homogeneous solution was freeze-dried to obtain the HClsalt of the product as a white soild (380 mg, quantitative). Thestructure was in agreement with its LC/MS and ¹H NMR data. ¹H NMR (D₂O)δ 4.57-4.40 (m, 1H), 3.59 (t, 6.6 Hz, 1H), 3.58 (s, 2H), 3.52-3.25 (m,5H), 3.15 (brs, 1H), 2.59-2.51 (m, 2H), 2.45-2.32 (m, 2H), 2.10-1.59 (m,2H), 1.45 (brs, 6H).

Example 35 3-[1-(Carboxymethylcarbamoyl)ethylsulfanyl]-2-hydroxyacrylicacid ethyl ester

[0790] 35A. Formula Ia where A is N-(propionyl)glycine, X is S, and Z is—O-Ethyl

[0791] A solution of N-(2-mercaptopropionyl)glycine (1.17 g, 7.17 mmol)and ethyl bromopyruvate (1 mL, 7.17 mmol, 90% purity) in acetonitrile(30 mL) was stirred at room temperature for 3 h. After removal of thesolvent, the residue was dissolved in a mixed solvent of ethanol (15 mL)and water (15 mL). After the pH was adjusted to 7-8 with satd. NaHCO₃solution, the mixture was stirred at room temperature overnight. Themixture was acidified to pH 1-2 with concentrated hydrochloric acid andthen rotary evaporated to dryness. The residue was purified by columnchromatography eluted with EtOAc giving3-[1-(carboxymethylcarbamoyl)ethylsulfanyl]-2-hydroxyacrylic acid ethylester as yellow solid (1.06 g, yield 53.3%). ¹H NMR (CDCl₃, 300.16 MHz)δ (ppm): 9.10 (broad s, 1H), 7.22 (d, J=0.9 Hz, 1H), 4.62 (d, J=17.7 Hz,1H), 4.53 (d, J=17.7 Hz, 1H), 4.28 (q, J=7.1 Hz, 2H), 3.47 (qd, J=6.2 &0.9 Hz, 1H), 1.48 (d, J=6.2 Hz, 3H) and 1.30 (t, J=7.1 Hz, 3H). ¹³C NMR(CDCl₃, 75.48 MHz) δ (ppm): 174.02, 165.84, 160.57, 130.27, 120.20,62.04, 46.76, 37.11, 14.45 and 14.09. MS (ESI) m/z: 260 (M+−OH, 100).

[0792] 35B. Formula Ia where A is 2,3-Dihydroxy-propyl, X is S. and Z is—O-Ethyl

[0793] Similarly, by following the procedure of Example 35A andsubstituting N-(2-mercaptopropionyl)glycine with3-mercapto-1,2-propanediol, there is obtained the following compound:

[0794] 3-(2,3-dihydroxypropylsulfanyl)-2-hydroxyacrylic acid ethylester. ¹H NMR (CDCl₃, 300.16 MHz) δ (ppm): 6.70 (d, J=1.1 Hz, 1H), 4.23(q, J=7.1 Hz, 2H), 4.17-4.11 (m, 1H), 3.82 (d, J=5.1 Hz, 2H), 2.99-2.88(m, 2H) and 1.30 (t, J=7.1 Hz, 3H). MS (ESI) m/z: 205 (M⁺−OH, 100), 227(M⁺−H₂O+Na, 51) and 431 [2(M⁺−H₂O)+Na, 29].

Example 36 2-Oxo-3-(4-oxo-3,4-dihydro-quinazolin-2-ylsulfanyl)-propionicacid ethyl ester

[0795] An homogeneous solution of 2-mercapto-3H-quinazolin-4-one (1.30g, 7.17 mmol) and ethyl bromopyruvate (1 mL, 7.17 mmol, 90% purity) inDMF (30 mL) was stirred at room temperature for 6 h. Triethylamine (5mL) was added to the solution and the mixture was stirred at roomtemperature for 36 h. The mixture was acidified to pH 1-2 withconcentrated hydrochloric acid and then rotary evaporated to dryness.The residue was separated by column chromatography eluted with EtOAcgiving the title compound as yellow solid (0.231 g, yield 11.0%). ¹H NMR(DMSO-d₆, 300.16 MHz) δ (ppm): 8.05 (dd, J=7.9 & 1.4 Hz, 1H), 8.01 (s,1H), 7.81 (td, J=8.3 & 1.5 Hz, 1H), 7.53 (d, J=8.2 Hz, 1H), 7.46 (td,J=7.5 & 1.4 Hz, 1H), 4.21 (q, J=7.1 Hz, 2H), 3.82 (d, J=12.2 Hz, 1H),3.46 (d, J=12.2 Hz, 1H) and 1.18 (t, J=7.1 Hz, 3H). ¹³C NMR (DMSO-d₆,75.48 MHz) δ (ppm): 168.06, 159.54, 159.26, 149.07, 135.98, 126.85,126.74, 126.36, 119.09, 91.33, 62.79, 37.92 and 14.31. MS (ESI) m/z: 293(M⁺+H, 100).

Example 37 3-(Benzoselenazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester

[0796] To a suspension of 2-mercaptobenzselenazole (418 mg, 2 mmol) in 4mL of MeCN and 4 mL of dichloromethane with vigorous stirring was addeddropwise, ethyl bromopyruvate (290 mg, 2 mmol). The resulting mixturewas stirred for 5 h. It was allowed to settle for 30 min and thenfiltered. The solid was washed with EtOAc/hexanes (1:1, 2×8 mL) anddried in high vacuum to afford3-(benzoselenazol-2-ylsulfanyl)-2-oxo-propionic acid ethyl ester as awhite powder (582 mg, 89%). ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm): 8.14-8.10(m, 1H), 7.95-7.92 (m, 0.7H), 7.75-7.73 (m, 0.3H), 7.52-7.45 (m, 1H),7.36-7.30 (m, 1H), 7.07 (s, 0.7H), 4.69 (s, 0.6), 4.37-4.27 (m, 2H),1.32 (t, J=7 Hz, 3H). MS(ESI) m/z: 330(M+H⁺, 55), 348 M⁺+H₂O, 58), 362(M⁺+MeOH, 100).

Example 38 Determination of Activity Utilizing Neuronal Cell StressAssay

[0797] A. Isolation and Culture of Primary Hippocampal Neuronal Cells.

[0798] The following materials are employed:

[0799] Neurobasal/B27i: Neurobasal medium (available from Invitrogen,San Diego, Calif.) with 1x B27 supplement (Invitrogen), 0.5 μML-glutamine, 25 μM L-glutamic acid, and 1× Penicillin/Streptomycin.

[0800] Hank's Basic Salt Solution (HBSS, Ca/Mg-free) is prepared bypreparing 1× Hanks CMF (Gibco) supplemented with HEPES (10 mM, pH 7.3),sodium bicarbonate (0.35%), 1× Penicillin/Streptomycin, and 1 mM MEMsodium pyruvate.

[0801] Poly-D-lysine (Sigma, St. Louis, Mo.), 50 μg/ml solution.

[0802] Sigmacote (Sigma, St. Louis, Mo.).

[0803] Plastic Culture Flasks (T75 cm²) or 24-well cell culture platestreated with Poly-D-Lysine (Sigma, St. Louis, Mo.).

[0804] A pregnant female mouse (E18-E19) is euthanized with CO₂ followedby removal of the uterus, which is then placed in a sterile plasticpetri dish. The embryos are removed from the sac, and the embryonicbrains removed and immersed in cold (4° C.) Buffered Salt Solution(HBSS; Ca/Mg free; Life Technologies) in a small petri dish. Hippocampiare then removed from the brains under a dissecting microscope andplaced on a paraffin-covered dish. The meninges are stripped away andthe dissected hippocampi are collected in a small petri dish in HBSS.The hippocampi are transferred to a 15-ml centrifuge tube (normally10-12 brains) filled with HBSS. The tube containing the brains iscentrifuged at 1000 rpm for 2 min in a tabletop centrifuge. Thesupernatant is removed, 2 ml of HBSS is added to the hippocampi in thetube, and the resulting suspension is triturated 2 times each withlong-tipped siliconized glass pipettes having progressively smallerapertures, starting with a pipette with a standard size opening(approximately 1.0 mm diameter), following with one having an apertureof half standard size (approximately 0.5 mm diameter), then with onehaving an aperture about one-half that size (0.25 mm diameter). Thesuspension is then centrifuged again at 1000 rpm for 2 min in a tabletopcentrifuge, the supernatant is discarded, and 2 ml of Neurobasal/B27i(with antibiotics) is added to the tube. The trituration proceduredescribed above is then repeated on this suspension.

[0805] The density of cells is determined on a small aliquot of cellsusing standard counting procedures and correcting for cell viability bytrypan blue stain exclusion. Using this procedure, the expected yield is3×10⁵-6×10⁵ cells/brain. Cells are then added to PDL-coated 24 wellplates, flasks or MetTek dishes in Neurobasal/B271 at a density of about1.5×10⁶ cells (T75 flask) or about 100,000 cells/well of a 24-wellplate. Plated cells are incubated at 37° C. in an atmosphere of 5%CO₂/95% O₂. Media is renewed after 3-4 days by replacing half of it withfresh Neurobasal/B27m medium, containing 5 μM cytosine arabinoside(Ara-C). Seven to eight days from the initial culture, the media isrenewed again, by removing one-half or it and replacing with an equalamount of fresh Neurobasal/B27m medium (without Ara-C).

[0806] B. Hippocampal Anoxia-Reoxygenation Cell Death Assay.

[0807] This assay is used to induce ischemia by anoxia-reoxygenation incultured hippocampal neuronal cells. Test compounds are added to assesspotency and efficacy against ischemia-induced neuronal cell injury andcell death.

[0808] The following materials are employed:

[0809] Neurobasal media, NoG neurobasal media, B27 supplement and B27Supplement minus AO (Invitrogen).

[0810] Neurobasal/B27 medium is prepared with 2× B27 minus AOsupplement, 0.5 mM L-glutamine and 0.25× penicillin/streptomycin.

[0811] Cell Tracker Green was obtained from Molecular Probes and a fresh5 μM solution was prepared from 10 mM stock just before use.

[0812] NoG-Neurobasal contains NoG neurobasal medium plus 0.5 mMglucose, 0.1 mM L-glutamine and 0.25× Penicillin/Streptomycin.

[0813] Primary hippocampal neuronal cells were prepared according to themethods described above and were cultured in poly-D-lysine coated 24well plates for 10-11 days prior to use.

[0814] Deoxygenated LoG-Neurobasal medium (100 ml) is prepared bypre-equilibrating the medium in a T150 cm² flask in a hypoxic chamberovernight. Following pre-incubation under hypoxic conditions, theLoG-Neurobasal media is lightly bubbled with 100% N₂ for 30 min tocompletely deoxygenate the media. An additional 20 ml LoG-Neurobasal ispre-equilibrated in a T75 cm² flask and 100 ml Neurobasal/B27AO isincubated in a normal incubator (5% CO₂) overnight. Reoxygenated mediumis prepared by placing medium overnight in the culture incubator (5%CO₂/95% O₂) prior to use.

[0815] Existing culture medium (Neurobasal/B27m) is removed from thecells by aspiration. Cells are washed once with 2 ml/well (24-wellculture plates) of glucose free-BSS. Neurons are replenished 10-11 daysafter initial culture with deoxygenated LoG-Neurobasal (1 ml per wellfor each well of a 24-well plate). Test compounds are added directly toeach well (3 concentrations of the compound plus positive control, eachin triplicate). Most test compounds are dissolved in 100% DMSO;concentrations are adjusted such that the final concentration of DMSO inthe cell media never exceeded 0.5%. Plates containing cells with testcompounds are placed in a hypoxic chamber for 5 hr with plate lids ajar.For normoxia controls, pre-equilibrated normoxic LoG-Neurobasal mediumis added to each well of cells, and the plate is replaced in the normalculture incubator for 5 hr. After 5 hr of hypoxia, the existing media iscarefully aspirated off, and 2 mL of new, reoxygenated(pre-equilibrated) Neurobasal/B27AO is added to each well. The same testcompounds (in the same the concentrations) are added back into thecorresponding wells. Plates are placed in the cell culture incubator (5%CO₂/95% O₂) and reoxygenated for 20-24 hr. After reoxygenation for 20-24hr, live neurons are quantitated using the cell tracker greenfluorescence method, described below.

[0816] To test for cell viability, existing culture medium is aspiratedfrom each well of the 24 well plates, and neurons are washed once with 2ml of HBSS (pH 7.4, prewarmed to 30-37° C.). To each well is added onemilliliter of 5 μM Cell Tracker Green fluorescent dye dissolved in HBSS.Plates are placed in the dark at room temperature for 15 minutes, andare then washed with two milliliters of HBSS. One milliliter of HBSS isthen added to each well, and fluorescent cells are counted using afluorescent microscope. Significantly increased cell viability comparedto control cells is indicative of a protective compound.

[0817] Results

[0818] When tested as described above, compounds of the presentinvention, including:

[0819]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0820]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0821]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]butyricacid,

[0822]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]butyricacid,

[0823]2-amino-4-[2-(2-butoxycarbonyl-2-methoxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0824]2-amino-4-[2-(2-benzyloxyimino-2-butoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0825]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0826]2-amino-4-[2-(2-benzyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl slat thereof,

[0827]2-amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-ethoxycarbonyl-2-(4-nitro-benzyloxyimino)-ethylsulfanyl]-ethylcarbamoyl}-butyricacid,

[0828]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[3-(4-methyl-piperidin-1-yl)-2,3-dioxo-propylsulfanyl]-ethylcarbamoyl}-butyricacid,

[0829]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hydroxyimino-3-oxo-3-piperidin-11-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0830]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-diethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0831]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0832]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(1-methoxycarbonyl-2-phenyl-ethylcarbamoyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid,

[0833]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-cyclohexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0834]2-Amino-4-[2-(2-benzylcarbamoyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid,

[0835]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(3-morpholin-4-yl-2,3-dioxo-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0836] 3-(1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0837] 3-(5-nitro-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester, and

[0838] 3-(5-methoxy-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester,

[0839] provided protection against stressor-induced cell death in atleast about 30% up to about 85% of the cells tested, at concentrationsranging from about 1 to about 350 μM.

Example 39 Myocyte Calcium-Contractility Assay.

[0840] A. Isolation and Culture of Primary Neonate Myocytes.

[0841] The following materials are employed:

[0842] 10× Heart Dissection Solution (HDS) contains the followingcomponents (g/l) in tissue grade water: NaCl, 68; HEPES, 47.6; NaH₂PO₄,2; Glucose, 10; KCl, 4; MgSO₄, 1, pH adjusted to 7.4. Prior to filtersterilization of diluted (1× HDS) solution, 10 mg phenol red is added toeach 500 milliliters of medium.

[0843] Transferrin and Bovine Insulin (available from Life Technologies)are resuspended at a concentration of 4 mg/ml in tissue culture gradewater.

[0844] DMEM-F12-DMEM/F12, powder, 1:1 containing glutamine andpyridoxine hydrochloride (available from Life Technologies). To oneliter equivalent of the powder is added 2.43 g of sodium bicarbonate and10 ml of 10OX Penicillin/Streptomycin in 950 ml of tissue culture gradewater with stirring. The pH is adjusted to 7.2 with 1 M HCl and volumeadjusted to 1 liter. The solution is filter sterilized, followed by theaddition of 2.5 ml of 4 mg/ml Transferrin, 250% 4 mg/ml Insulin and 30.7mg of bromodeoxyuridine.

[0845] DMEM-F12-5% FBS is also prepared for pre-coating the tissueculture plates and initial suspension of the cardiomyocyte pellet.

[0846] Collagenase solution-57.1 mg of collagenase is resuspended in 140ml 1× HDS.

[0847] Tissue culture ware is pre-coated with DMEM-F12-5% FBS byincubating 50 μl per well of a 96-well plate and 0.5 ml per 24-wellplate at 37° C.

[0848] Two-day old rat pups are removed from their mothers and placed ina sterile container. Pups are dipped quickly into 70% alcohol, thendecapitated and the body placed in an empty sterile tissue culture dish.An incision is made starting at the neck and progressing towards thebelly, cutting through the sternum. The heart is removed and placed in atissue culture dishes containing 1× HDS. The atria are trimmed, and theremaining ventricles are placed into a separate tissue culture dishcontaining 1× HDS, where they are sectioned into 3-4 pieces each.Ventricles are then transferred to a sterile 250 ml glass flask and the1× HDS is removed. Twenty milliliters of pre-warmed collagenase solutionis added to the ventricles, followed by incubation at 37° C. withshaking. After 20 minutes, the collagenase solution is removed andreplaced with 20 ml fresh pre-warmed collagenase. Incubation iscontinued for an additional 20 minutes. At the end of the incubation,any tissue chunks are allowed to settle prior to removing thecollagenase (containing the isolated cardiomyocytes) from the disruptedtissue pieces. The isolated myocytes are added to a 50 ml Falcon tubecontaining 2 ml Fetal Bovine Serum (FBS). The remaining tissue piecesare subjected to a second digestion by adding 20 ml fresh pre-warmedcollagenase and incubating as above for 20 minutes. This second digestis then centrifuged at 1000 rpm for 5 minutes (tabletop centrifuge). Theresulting supernatant is discarded, and the cell pellet is suspendedwith 4 ml FBS. The resulting cell suspension is placed in the incubatorat 37° C. This step is repeated several additional times to harvestadditional material.

[0849] Percoll gradients are prepared by adding 2.5 ml of lOx HDS to22.5 ml of Percoll (Life Technologies) with mixing (Percoll Stock). TopGradient solution (11 ml Percoll Stock and 14 ml 1× HDS) and BottomGradient solution (13 ml Percoll Stock and 7 ml 1× HDS) are prepared.Four milliliters of the Top Gradient solution is transferred into 6×15ml sterile Falcon tubes. Three milliliters of the Bottom Gradientsolution is placed in each tube by inserting a serological pipette tothe bottom of the tube and slowly adding the liquid.

[0850] All the digests (6) are pooled in one 50 ml Falcon tube andcentrifuged on a tabletop centrifuge at 1000 rpm for 10 minutes. Thesupernatant is discarded, and the cell pellet is resuspended in 12 ml of1× HDS. Two milliliters of the cell suspension is added to the top ofeach gradient. The gradient tubes are then centrifuged at 3000 rpm for30 minutes without braking in a Beckman Allegra 6 centrifuge (GH 3.8Arotor). Following centrifugation, the cells segregate into two sharpbands at the two interfaces. The lower band of the two bands is enrichedfor cardiomyocytes; there is also a cardiomyocyte pellet at the bottomof the tube. The upper band is enriched for fibroblasts and othernon-cardiomyocytes. The upper portion of the gradient is aspirated downto just above the cardiomyocyte layer. The cardiomyocyte layer is thencarefully removed along with the pellet, and the two fractions arepooled in a sterile 50 ml Falcon tube, along with correspondingfractions from additional gradient tube; then 1× HDS is added to a totalvolume of about 50 ml. The tube is centrifuged at 1000 rpm for 10minutes. The supernatant is discarded and resuspended in 10 ml 1× HDS. Afurther 40 ml of 1× HDS is added and the centrifugation step isrepeated. The cell pellet is resuspended carefully but thoroughly in 50ml of DMEMF12-5% FBS.

[0851] A small aliquot of the cell suspension is counted in ahemocytometer. The DMEMIF12-FBS coating medium is aspirated from thetissue culture dishes. The cardiomyocytes are added to the dishes at aplating density of 7.5×10⁴/well per 96-well in 200 μL and 6×10⁴/well per24-well in 1 ml. The cultures are incubated at 37° C. with 5% CO₂overnight. The original medium is removed, and add fresh DMEM/F12-5% FBSis added to each culture, prior to incubation at 37° C. with 5% CO₂ fora further 48 hours, before use.

[0852] B. Contractility Assay

[0853] The following materials are employed:

[0854] Complete DMEM-F12: DMEM/F12, powder, 1:1 containing glutamine andpyridoxine hydrochloride (available from Life Technologies—InvitrogenLife Technologies, Carlsbad, Calif.). Powder sufficient to prepare oneliter of buffer and 2.43 g of sodium bicarbonate is mixed into 950 ml oftissue culture grade water. The pH is adjusted to 7.2 with 1 M HCl andthe remaining water added to make 1 liter. Following filtersterilization, 1 0 ml of 100× Penicillin/Streptomycin, 2.5 ml of 4 mg/mlTransferrin, 250 μl 4 mg/ml Insulin and 30.7 mg of bromodeoxyuridine areadded, and the mixture is incubated at 37° C. prior to use.

[0855] 1 mM glucose in DMEM is made from DMEM without L-glutamine,without glucose, without sodium pyruvate (available from LifeTechnologies).

[0856] 20 μM Fluo-4: Cell permanent AM ester of Fluo-4 (available as adry powder to be stored at −20° C., from Molecular Probes—Eugene,Oreg.). This fluorescent dye is light sensitive and should be made upfresh at 1 mM in DMSO prior to use to prevent light degradation.

[0857] Neonatal cardiomyocytes are isolated as described above. Thecardiomyocytes are plated in 96-well format (black clear-bottomedplates) at a density of 7.5×10⁴ per well and grown for 2 days in thepresence of 5% FBS prior to use in the assay.

[0858] Physiological ischemia is simulated by placing the cardiomyocytesin an anaerobic chamber (0% O₂, 85% N₂, 5% CO₂ & 10% H₂) in DMEMcontaining 1 mM glucose. Positive control cells are treated withDMEM-F12 containing 25 mM Glucose, which protects against the anoxia.

[0859] The test compounds are made up in DMEM-1 mM glucose in 96deep-well mother plates and appropriately diluted for use in the assay.The media is removed from the cells and replaced with 200% of eitherDMEM-F12 or 1 mM DMEM with or without test compounds. The plates arethen placed inside a 37° C. incubator in the anaerobic chamber andincubated for 16 hours. The plates are then removed and reoxygenated bythe addition of pre-warmed DMEM-F12 containing 5% FBS. Since the anoxictreatment may damage and/or kill the cells, causing them to dislodgefrom the bottom of the wells gentle aspiration of media is required atthis step. The cells are then placed in a normal incubator at 37° C. andincubated for two hours to allow the cells to reoxygenate.

[0860] A working solution of 20 μM Fluo-4 is added to pre-warmed 1×HBSS. The cells are loaded with Fluo-4 by first removing media from thecells and replacing with 100 μl of 20 μM Fluo-4. Unloaded control cellsare treated in parallel with 1× HBSS alone. All cells are then incubatedat 37° C. for 30 minutes. Before fluorescence measurements are made, thecells are washed in indicator-free medium (HBSS) to remove any dye thatis non-specifically associated with the cell surface. Cells are thenincubated for an additional 20 minutes at room temperature. Basal Fluo-4fluorescence is measured using the 485 nm excitation and 538 nm emissionfilter pair on a microplate flourometer (Fluorskan™, Thermo LabsystemsOy, Helsinki, Finland). Each well is read for 60 ms to obtain a baselinereading, then removed from the fluorimeter and stimulated to contract byaddition of 1× HBSS (which contains 1.3 mM CaCl₂), followed byincubation at 37° C. for 90 minutes. A second fluorescence reading isthen taken. Difference in pre vs. post stimulation fluorescence readingsis indicative of activity.

[0861] Results

[0862] When tested as described above, compounds of the presentinvention, administered at concentrations of about 100 μM to about 1000μM, including:

[0863]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-oxo-2-pentyloxycarbonyl-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0864]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-[ethylcarbamoyl]-butyricacid,

[0865]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0866]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(3-morpholin-4-yl-2,3-dioxo-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0867] 3-(1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0868] 3-(5-chloro-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0869] 3-(5-nitro-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester, and

[0870] 3-(5-methoxy-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester,

[0871] showed the presence of calcium transients in about 25 to about90% of the cells, with amounts indicative of ability guard againstischemic damage and allow the cells to maintain their contractilefunction.

Example 40 Rat Middle Cerebral Artery Occlusion (MCAO) Model of CerebralIschemia

[0872] A. Animal Preparation

[0873] Male Wistar rats (Harlan, Ind.) weighing 300-350 g are commonlyused in these experiments. Animals are allowed free access to water andcommercial rodent diet under standard laboratory conditions. Roomtemperature is maintained at 20-23° C. and room illumination is on a12/12-hour light/dark cycle. Animals are acclimatized to the laboratoryenvironment 5 to 7 days prior to the study, and fasted (with free accessto water) overnight before surgery.

[0874] B. Middle Cerebral Artery Occlusion (MCAO)

[0875] Anesthesia is maintained by inhalation of 3.0% isoflurane(Aerrane, Front Dodge, Iowa) in 0.8% oxygen. The animal's neck is shavedand sterilized before operation. Body temperatures are controlled andmaintained at 37.5° C.+/−1 degree via external heating and coolingdevices. To lower the body temperature, animals are placed in a coolingchamber that uses ice to cool circulating air. Throughout the study thebody temperature is recorded using a temperature transponder (BMDS Inc.,Seaford, Del.) implanted subcutaneously at the time of MCAO between therat shoulder blades, which allows the user to read the body temperaturevia a pocket scanner (BMDS Inc., Seaford, Del.). The body temperaturecan also be taken by inserting the temperature probe into the animal'srectum. Body temperature is recorded every hour for 6 hourspost-occlusion, but temperature is measured more frequently tofacilitate maintaining the animals' normothermic temperature.

[0876] Animals are subjected to two hours MCAO using a modifiedintraluminal filament technique, as follows. A midline incision on theventral part of the neck is made to expose external and internal carotidarteries. The right external and common carotid arteries are ligated bya suture (silk 5/0, Carlisle Laboratories, Farmers Branch, Tex.) and theright internal artery is temporarily ligated using a microvascular clip(Fine Science Tool Inc., Foster City, Calif.). A small incision is madein the common carotid artery. A nylon filament, its tip rounded byheating, is prepared from a fishing line (Stren Fishing Lines,Wilmington, Del.) and is inserted from the right common carotid artery.The filament is advanced into the internal carotid artery 18-20 mm fromthe point of bifurcation of internal and external arteries and a sutureis tightly ligated around the filament. Two hours post occlusion,animals are re-anesthetized to allow reperfusion for the remaining ofthe experiment by removal of the filament. C. Drug Administration

[0877] Test compounds can be administered by any of a number of routes,such as those described below. Compounds can be administered before,during or after occlusion, as appropriate to the protocol.

[0878] a) Intracerebroventricular (ICV) Infusion

[0879] The anesthetized animal is placed on a stereotaxic apparatus(Harvard Apparatus, S. Natick, Mass.). Anesthesia is maintained byinhalation of 3.0% isoflurane (Aerrane, Front Dodge, Iowa) in 0.8%oxygen throughout the entire procedure. The scalp is shaved andsterilized prior to surgery. A midline sagittal incision about 3 cm longis made slightly behind the eyes to expose the skull. The skull isscraped with a rounded end spatula to remove periosteal connectivetissue. A bur hole is placed 1.5 mm lateral, 1 mm posterior to the leftof the bregma to mark the left lateral ventricle. A brain infusioncannula (ALZET—Alza, Palo Alto, Calif.) is inserted 4 mm deep into thehole. The desired depth is adjusted by attaching spacers to the cannula.The cannula, attached to a 4-cm silastic catheter (Helix Medical Inc.,Carpinteria, Calif.), is fixed in place with dental cement(Ketac-cement, Norristown, Pa.). The catheter is either attached to aprimed osmotic pump placed subcutaneously between the shoulder bladesfor permanent infusion or to a syringe for a short infusion.

[0880] b) Intravenous (IV) Osmotic Pump Implantation into the JugularVein

[0881] Anesthesia is maintained by inhalation of 3.0% isoflurane(Aerrane, Front Dodge, Iowa) in 0.8% oxygen throughout the entireprocedure. The animal's neck is shaved and sterilized before operation.A midline incision is made on the ventral part of the neck to exposesthe jugular vein. The vein is isolated and ligated with a suture (silk5/0, Carlisle Laboratories, Farmers Branch, Tex.) rostral to the pointof the incision and a microvascular clip (Fine Science Tool Inc., FosterCity, Calif.) is placed close to the heart. A small incision is madebetween the two ligations. A 2-cm silastic catheter (Helix Medical Inc.)attached to a PE-60 tube (Becton. Dickinson and Co. Sparks, Md.)connected to an ALZET (Alza, Palo Alto, Calif.) pump is introduced andadvanced 2 mm into the jugular vein toward the heart. The microvascularclip is removed and the catheter is secured in place with a suture (silk5/0, Carlisle Laboratories, Farmers Branch, Tex.). The pump is placedinto a pocket made subcutaneously between the shoulder blades, allowingthe catheter to reach over neck to the jugular vein with sufficientslack to permit free movement of neck and head.

[0882] c) IV Infusion Via Femoral Vein

[0883] Anesthesia is maintained by inhalation of 3.0% isoflurane(Aerrane, Front Dodge, Iowa) in 0.8% oxygen throughout the entireprocedure. The exterior site of the right femoral vein is shaved andsterilized prior to surgery. A 3-cm incision is made in the right groinregion and the femoral vein is isolated. A small incision is made on thefemoral vein, temporarily ligated with a microvascular clip, tointroduce and advance a polyethylene (PE-50) catheter (Becton Dickinsonand Co. Sparks, Md.). The catheter is secured in place with suture (silk5/0, Carlisle Laboratories, Farmers Branch, Tex.). The other end of thecatheter is attached to a syringe filled with the heparinized saline forthe bolus injection. Using a hemostat, a pocket is made subcutaneouslyon the back of the animal so the PE catheter can be brought up to theexteriorization point at the nape of the neck for either a bolusinjection or a continuous injection by an osmotic pump.

[0884] d) Intraperitoneal (IP) Injection

[0885] An awake rat is held in a standard hand hold position, a 23 3/4Gneedle is injected into the lower right quarter of the abdomen past theperitoneum, slightly off the midline. To avoid organ injection, theplunger of the syringe is slightly pulled back. If no fluid iswithdrawn, the content of the syringe is delivered into the abdominalcavity.

[0886] e) Gavaqe Feeding

[0887] A standard rat gavage tube (Popper & Sons Inc., NY) is attachedto a 3-cc hypodermic syringe. The animal is held by the shoulder in avertical position. The feeding tube is placed into the mouth thenadvanced until it reaches the stomach (the approximate insertion lengthof the tube was measured prior to the feeding). The content of thesyringe is slowly delivered, and then the tube is withdrawn.

[0888] D. Behavioral Assessment

[0889] One hour after MCAO, the animal is gently held by its tail andobserved for forelimb flexion. Then the animal is placed on the floor tobe observed for walking pattern; only the animals that score 3 on theBederson grading system (Table 1) are included in the study. TABLE 1Bederson Grading System for Neurological Evaluation Neurological deficitGrading Behavioral observation Normal grade 0: No observable deficitModerate grade 1: forelimb flexion Severe grade 2: forelimb flexion,decreased resistance to lateral push grade 3: forelimb flexion,decreased resistance to lateral push, circle to paretic side

[0890] E. Evaluation of Ischemic Damage

[0891] Twenty-four hours post-MCAO, or longer in some experiments,animals are sacrificed by CO₂ asphyxiation (dry ice). The brain isquickly removed from the skull, using standard procedures, rinsed inchilled saline solution, and placed on a rat brain tissue slicer (ASIinstrument, MI). Seven 2-mm thick coronal slices are cut from each brainusing razor blades. The slices are immersed in 0.9% saline containing1.0% 2,3,5-triphenyltetrazolume chloride (TTC) (Sigma Chemical Co., St.Louis, Mo.) and incubated in a 37° C. water bath for 30 minutes.

[0892] After staining, each 2-mm slice is photographed with a TMC-7camera (JH Technologies, Ca) which is directly connected to a desktop PCto capture and save the image of each brain slice. This image is usedfor the measurements of the regions of interest using a computer-basedimage processing system (Metamorph).

[0893] To measure each area, the region of interest is selected using afreehand selection tool, the area is automatically computed by selectingthe measure command. The measurements for primary regions of interestare right hemisphere, left hemisphere, total infarct, subcorticalinfarct, total penumbra and subcortical penumbra. After all regions ofinterest are measured for all seven slices of the brain, they are sortedby slice number and the corresponding regions of interest using anExcell macro called statistic final. This macro also calculates thecortical penumbra, cortical infarct and total ischemic damage for eachslice; the corresponding areas of each rat brain are added together toproduce a single measurement for each area. Since the ipsilateralhemisphere is swollen following MCAO, edema volume is calculated andreported as the volumetric differences between the right and lefthemispheres of each brain slice. Using the % of hemispheric swelling allthe volumes are corrected for the edema.

[0894] The volume of the damage is determined using the calculationsbelow for each rat's brain. Measurement Equation Cortical TotalCorrected Value(s) Penumbra (C.P.) Penumbra- Suboortical Total Penumbra(T.P._(corr)) = Penumbra (T.P. × % H.S./100) C.P._(corr.) = C.P.-(C.P. ×% H.S./100) S.P._(corr) = S.P.-(S.P. × % H.S./100) Cortical InfarctTotal Infarct- T.I._(corr.) = T.I.-(T.I. × Subcortical Infarct %H.S./100) S.I._(corr.) = S.I.-(S.I. × % H.S./100) C.I._(corr.) =C.I.-(C.I. × % H.S./100) Total Ischemic Total Penumbra +T.I.D._(corrected) = T.I.D.- Damage (T.I.D.) Total Infarct (T.I.D. × %H.S./100) Total Volume Each value is (mm³) multiplied by 2 (thethickness of the tissue). Edema Volume The volumetric differencesbetween the sum of right and left hemispheres determines the edemavolume. % Hemispheric Edema × 100/left swelling (H.S.) hemisphere

[0895] F. Statistical Analysis

[0896] Sample size is chosen to achieve a 90% probability of significantresults. The measurements representing the same region of interest inseven slices of each rat's brain are added together to yield a singlemeasurement for total infarct, subcortical infarct, cortical infarct,total penumbra, subcortical penumbra, cortical penumbra, total ischemicdamage and edema in each animal. Group data are presented as means+/−SEM. Differences at the level of p<0.05 are considered statisticallysignificant. Between groups, comparisons of each region of interest arecarried out by unpaired student t test (between two groups) or one wayANOVA followed by post hoc Bonferroni's multiple comparisons or by thenonparametric Dunnett's test (between control and the drug treatedgroups).

[0897] Results

[0898] When tested as described above, compounds of the presentinvention, including:

[0899]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0900]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]butyricacid,

[0901]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]

[0902] butyric acid,

[0903]2-amino-4-[2-(2-butoxycarbonyl-2-methoxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0904]2-amino-4-[2-(2-benzyloxyimino-2-butoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0905]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0906]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid

[0907]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hydroxyimino-3-oxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0908]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0909]2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(1-methoxycarbonyl-2-phenyl-ethylcarbamoyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid,

[0910] 3-(1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0911] 3-(5-nitro-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0912] 3-(5-methoxy-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester, and

[0913] 3-(4,5-dihydro-thiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester,

[0914] provided a reduction in total infarct volume of at least about20% up to about 80% at doses in the range of less than about 1 μg/kg toless than about 10 mg/kg.

Example 41 Model of Myocardial Infarction: Left Coronary Ligation (Rat)

[0915] Male Sprague-Dawley weighing 250-320 g are allowed free access towater and commercial rodent diet under standard laboratory conditions.Room temperature is maintained at 20-23° C. and room illumination is ona 12/12-hour light/dark cycle. Animals are acclimatized to thelaboratory environment 5 to 7 days prior to the study and are fastedovernight prior to surgery.

[0916] Surgical Procedure for Acute Studies:

[0917] Rats are anaesthetized with Urethane (1.2-1.5 gm/kg). Core bodytemperature is maintained at 37° C. by using a heating blanket. Thesurgical area is shaved, and a ventral midline incision is made toexpose the trachea and jugular area. A catheter (PE50) is placed in thejugular for administration of compound and maintenance anesthesia. Thetrachea is incised and a 14-16-gauge modified intravenous catheter isinserted and tied in place as an endotracheal tube. The animal is placedin right lateral recumbency and initially placed on a Harvard ventilatorwith a tidal volume of 5-10 ml/kg. 100% O₂ is delivered to the animalsby the ventilator. ECG electrodes are placed to record a standard LeadII ECG. The surgical site is cleaned with alcohol swab, and a skinincision is made over the rib cage over the 4^(th)-5^(th) intercostalspace. The underlying muscles are dissected with care to avoid thelateral thoracic vein, to expose the intercostal muscles. The chestcavity is entered through the 4^(th)-5^(th) intercostal space, and theincision expanded to allow visualization of the heart. The pericardiumis opened to expose the heart. A 6-0 silk suture with a taper needle ispassed around the left coronary artery near its origin, which lies incontact with the left margin of the pulmonary cone, at about 1 mm fromthe insertion of the left auricular appendage. A piece of tubing isplaced over the suture to form an occluder. The coronary artery isoccluded for 30 minutes by sliding the tube towards the heart untilresistance is felt and holding it in place with a vascular clamp. TheECG is monitored for S-T changes indicative of ischemia. After 30minutes, the occluder is removed, leaving the suture in place. The ECGis monitored for the first 10 minutes of reperfusion. The rat istransferred to the pressure control ventilator for the remainder of theprotocol. The rats are ventilated by a small animal ventilator with apeak inspiratory pressure of 10-15 cm H₂O and respiratory rate 60-110breaths/min. The heart is allowed to reperfuse for 90 minutes.

[0918] Surgical Procedure for 24 Hour Study:

[0919] Rats are anaesthetized with Ketamine/Xylazine IP (95 and 5 mg/kg)and intubated with a 14-16-gauge modified intravenous catheter.Anesthesia level is checked every 15 minutes by toe pinch. Core bodytemperature is maintained at 37° C. by using a heating blanket. Thesurgical area is shaved and scrubbed. A ventral midline incision is madeto expose the jugular vein. A catheter (PE50) is placed in the jugularfor administration of compound and maintenance anesthesia. The animal isplaced in right lateral recumbency and initially placed on a ventilatorwith a tidal volume of 5-10 ml/kg H₂O or a pressure controlledventilator with a peak inspiratory pressure of 8-15 cm H₂O andrespiratory rate 60-110 breaths/min. 100% O₂ is delivered to the animalsby the ventilator. ECG electrodes are placed to record a standard LeadII ECG. The surgical site is cleaned with surgical scrub and alcohol. Askin incision is made over the rib cage over the 4^(th)-5th intercostalspace. The underlying muscles are dissected with care to avoid thelateral thoracic vein, to expose the intercostal muscles. The chestcavity is entered through 4^(th)-5th intercostal space, and the incisionexpanded to allow visualization of the heart. The pericardium is openedto expose the heart. A 6-0 silk suture with a taper needle is passedaround the left coronary artery near its origin, which lies in contactwith the left margin of the pulmonary cone, at about 1 mm from theinsertion of the left auricular appendage. A piece of tubing is placedover the suture to form an occluder. The coronary artery is occluded for30 minutes by sliding the tube towards the heart until resistance isfelt and holding it in place with a vascular clamp. The ECG is monitoredfor S-T changes indicative of ischemia. After 30 minutes, the occluderis removed, leaving the suture in place. The ECG is monitored for thefirst 10 minutes of reperfusion. The incision is closed in three layers.The IV catheter is removed or tunneled under the skin and exteriorizedbetween the shoulder blades to allow for blood withdrawal or furtherdrug therapy. The rat is ventilated until able to ventilate on its own.The rats are extubated and recovered on a heating pad. Once awake, theyare returned to their cage(s). Animals may receive Buprenorphine(0.01-0.05 mg/kg SQ) for post-operative analgesia. After the designatedreperfusion time (24 hours) the animals are anesthetized and the heartsremoved under deep anesthesia.

[0920] Treatment Protocols

[0921] Diet Animals are fed a custom diet prior to or after coronaryligation. The length of treatment varies with the study. Doses arecalculated based on the average consumption of 15 gms of feed per dayfor a 300 gm rat. Rat weights are monitored during the study. Feed notconsumed is weighed to estimate consumption rates.

[0922] Gavage

[0923] Animals are dosed orally by gavage. Length and frequency oftreatment vary with the study. A standard rat gavage tube (Popper & SonsInc, NY) is attached to a 3-cc hypodermic syringe. The animal is held bythe shoulder in a vertical position. The feeding tube is placed into themouth then advanced until it reaches the stomach (the approximateinsertion length of the tube is measured prior to the feeding). Thecontent of the syringe is slowly delivered, and then the tube iswithdrawn.

[0924] IV Treatment

[0925] A ventral incision is made to expose the jugular area. A catheter(PE50) is placed in the jugular vein for administration of compound.Animals are dosed by bolus injection and/or continuous infusion. Thetime and duration of treatment varies with the protocol.

[0926] Tissue Processing

[0927] After reperfusion, each animal receives 200 units of heparin IVunder general anesthesia and the heart is removed and placed in coldsaline. After removal the coronary artery is ligated with the suturethat is already in place. The heart is placed on a perfusion apparatusand Evans Blue dyed is infused delineate the area at risk. The heart isthen cut into five 2-mm thick transverse slices from apex to base. Theslices are incubated in 1% triphenyltetrazolium chloride (TTC) in 0.9%saline for 20 minutes at 37° C. Tetrazolium reacts with NADH in thepresence of dehydrogenase enzymes causing viable tissue to stain a deepred color and that is easily distinguished from the infarctedpale-unstained necrotic tissue. The slices are placed apex side down inthe lid of a small petri dish for the staining procedure. The bottom ofthe dish is placed over the slices to keep them flat. The slices arephotographed in order from apex to base, with the base side up. Theareas of infarcted tissue, area at risk and the whole left ventricle aredetermined using a computerized image analysis system. The total areafor each region is added together to give a total for the entire heart.Infarct size is expressed both as a percentage of the total ventricleand the area at risk.

[0928] Statistical Analysis

[0929] Group data is represented as means +/−SEM. Comparisons betweentreatment groups are made using ANOVA with p<0.05 consideredsignificant. Post hoc comparisons may be made using either Dunnett'stest or Tukey's test.

[0930] Results

[0931] When tested as described above, compounds of the presentinvention, including:

[0932]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]butyricacid,

[0933]2-amino-4-[2-(2-butoxycarbonyl-2-methoxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0934]2-amino-4-[2-(2-benzyloxyimino-2-butoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,

[0935]2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,

[0936]2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid, and

[0937] 3-(5-chloro-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethylester, show activity in the range of about 15% to about 55% infarct sizereduction.

Example 42 Evaluations of Sensorimotor Behavior

[0938] A. Fore and Hindlimb Grip Strength Test in Rats

[0939] Animals with cerebral infarction induced by transient orpermanent unilateral occlusion of the middle cerebral artery (MCA) andsham-operated rats are tested for grip strength, a standard model ofneuromuscular function and sensorimotor integration, using aComputerized Grip Strength Meter for Rats (Dual Stand Model, ColumbusInstruments, Columbus, Ohio).

[0940] Animals are moved into the testing room for 30 minutes beforetesting. Prior to testing, each gauge is calibrated with a set of knownweights and the apparatus is adjusted for the size of animal, accordingto manufacturer's instructions. The forelimb measurements are carriedout with the meter in the tension peak mode to freeze the reading as thesubject is pulled away from the grip bar. The hindlimb measurements arecarried out with the meter in the compression peak mode to freeze thereading as the subject's hindlimbs are pulled over the bar toward themeter. Each animal is hand-held by the investigator as pulled past thegrip bars, using a consistent technique, leaving the fore and hind limbsfree to grasp the grip bars.

[0941] Testing is carried out on postoperative day 2 and repeated, in ablind-randomized fashion, twice weekly for a defined interval.Typically, three successive readings are taken for each animal with anintertrial interval long enough to record the data and zero both metersfor the next trail.

[0942] B. Rota-Rod Test in Rats

[0943] Apparatus: Rota-Rod Treadmill for Rats (7750 Accelerating Model,from UGO BASILE, COMERIO-ITALY).

[0944] Procedure: Animals with cerebral infarction induced by transientor permanent unilateral occlusion of the middle cerebral artery (MCA)and sham-operated rats are tested in this study, using a Rota-RodTreadmill for Rats (7750 Accelerating Model, UGO Basile, Comerio,Italy). The animals are moved into the testing room 30 minutes beforetesting. Every rat receives 2-3 training runs of 1-2 minutes atintervals of 2-3 hours before testing.

[0945] The cylinder on the apparatus is set in motion before placing therats in position. The motor is set at a constant selected speed in 7700on RESET mode, and the rats are placed, one by one, in their sections.

[0946] Testing is carried out on postoperative day 2 and repeated, in ablind-randomized fashion, twice weekly for a defined interval.Typically, three successive readings are taken for each animal with anintertrial interval long enough to record the data and zero both metersfor the next trail.

[0947] The compounds of the present invention show activity when testedby this method.

Example 43 Model of Congestive Heart Failure

[0948] Experimental Preparation

[0949] 225-275 g male Sprague-Dawley CD (Charles River) rats are usedfor this experiment. Animals are allowed free access to water andcommercial rodent diet under standard laboratory conditions. Roomtemperature is maintained at 20-23° C. and room illumination is on a12/12-hour light/dark cycle. Animals are acclimatized to the laboratoryenvironment 5 to 7 days prior to the study. The animals are fastedovernight prior to surgery.

[0950] Animals are anaesthetized with ketamine/xylazine (95 mg/kg and 5mg/kg) and intubated with a 14-16-gauge modified intravenous catheter.Anesthesia level is checked by toe pinch. Core body temperature ismaintained at 37° C. by using a heating blanket. The surgical area isclipped and scrubbed. The animal is placed in right lateral recumbencyand initially placed on a ventilator with a peak inspiratory pressure of10-15 cm H₂O and respiratory rate 60-110 breaths/min. 100% O₂ isdelivered to the animals by the ventilator. ECG electrodes are placed torecord a standard Lead II ECG. The surgical site is scrubbed withsurgical scrub and alcohol. An incision is made over rib cage over the4^(th)-5^(th) intercostal space. The underlying muscles are dissectedwith care to avoid the lateral thoracic vein, to expose the intercostalmuscles. The chest cavity is entered through 4^(th)-5^(th) intercostalspace, and the incision expanded to allow visualization of the heart.The pericardium is opened to expose the heart. A 6-0 silk suture with ataper needle is passed around the left coronary artery near its origin,which lies in contact with the left margin of the pulmonary cone, atabout 1 mm from the insertion of the left auricular appendage. Thecoronary artery is occluded by tying the suture around the artery. TheECG is monitored for S-T changes indicative of ischemia. If the animaldevelops ventricular fibrillation, gentle cardiac massage is used toconvert the animal to a normal rhythm. The incision is closed in threelayers. The rat is ventilated until are able to ventilate on their own.The rats are extubated and recovered on a heating pad. Animals receivebuprenorphine (0.01-0.05 mg/kg SQ) for post operative analgesia. Onceawake, they are returned to their cage. Animals are monitored daily forsigns of infection or distress. Infected or moribund animals areeuthanized. Animals are weighed once a week.

[0951] Treatment Protocols

[0952] Diet

[0953] Animals are fed a custom diet prior to or after coronaryligation. The length of treatment will vary with the study. Doses arecalculated based on the average consumption of feed per day. Rat weightsare monitored during the study. Feed not consumed is weighed to estimateconsumption rates.

[0954] Gavage

[0955] Animals are dosed orally by gavage. Length and frequency oftreatment will vary with the study. A standard rat gavage tube (Popper &Sons Inc, NY) is attached to a 3-cc hypodermic syringe. The animal isheld by the shoulder in a vertical position. The feeding tube is placedinto the mouth then advanced until it reaches the stomach (theapproximate insertion length of the tube is measured prior to thefeeding). The content of the syringe is slowly delivered, and then thetube is withdrawn.

[0956] Drinking Water

[0957] Compound can also be dissolved in the drinking water. Waterconsumption is monitored. In the case of a bitter tasting compound,flavoring agents may be added to the water of both vehicle and treatedgroups. In the case of insoluble compounds, solubilizing agents may beused (i.e. 0.015% cremophor.0.015% alcohol).

[0958] Alzet Pumps

[0959] Alzet pumps can be implanted using aseptic techniques into theperitoneum or subcutaneously behind the shoulder blades. Pumps areimplanted using Isoflurane anesthesia. Serial implantation can be usedfor extended studies.

[0960] Measurements

[0961] In Vivo

[0962] After 6-12 weeks the animals are anesthetized withKetamine/Xylazine (95 mg/kg and 5 mg/kg), and a catheter is placed inthe right carotid artery and advanced into the left ventricle forhemodynamic measurements. The catheter is attached to a pressuretransducer calibrated against a mercury manometer immediately prior touse. Recordings are made by a DATAQ data analysis system. Pressuretraces are recorded and analyzed for heart rate, left ventricularsystolic and diastolic pressure, left ventricular developed pressure,and dP/dt max and min. An average of at least five peaks is used todetermine values for left ventricular systolic and end diastolicpressure. Left ventricular developed pressure is determined bysubtracting end diastolic pressure from left ventricular systolicpressure. Heart rate is determined from the frequency spectrum of a 5second sample. After measurements are taken, 2 ml blood is removed andplaced in serum and plasma tubes for possible analysis.

[0963] Ex Vivo

[0964] After removal, the heart is placed in cold saline to stop thebeating, then trimmed and weighed. Heart weight is presented as totalweight and as a percentage of total body weight. After removal of theheart, lungs and liver are weighed and dried overnight for determinationof wet to dry ratios.

[0965] The heart is sliced and slice #3 is incubated in 1%triphenyltetrazolium chloride (TTC) in 0.9% saline for 20 minutes at 37°C. Tetrazolium reacts with NADH in the presence of dehydrogenase enzymescausing viable tissue to stain a deep red color that is easilydistinguished from the infarcted pale-unstained necrotic tissue. Theslice is placed apex side down in the lid of a small petri dish for thestaining procedure. The bottom of the dish is placed over the slice tokeep it flat. The slice is photographed. The areas of infarcted tissue,left and right ventricle are determined using a computerized imageanalysis system. Infarct size is expressed as a percentage of the totalventricle. Total areas of the left and right ventricle are measured. Theremaining sections are divided into right and left ventricle and frozenfor TBARS and glutathione assays.

[0966] Statistical Analysis

[0967] Group data is presented as means +/−SEM. Comparisons betweentreatment groups are made using ANOVA with p<0.05 consideredsignificant. Post hoc comparisons use either Dunnett's test or Tukey'stest. Survival curves are generated using Graph Pad Prism. For each Xvalue (time) Prism shows the fraction still alive. It also showsstandard error. Prism calculates survival fractions using the productlimit or Kaplan-Meier method.

[0968] The compounds of the present invention, administered in thedrinking water in concentrations ranging from 10 mg/L to 1000 mg/L withtreatment initiated 1 week following ligation, show activity when testedby this method.

[0969] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

We claim:
 1. A method of treating a mammal having a conditioncharacterized by oxidative stress, comprising administering atherapeutically effective amount of a compound of Formula I:

wherein: A is: optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl, optionally substituted heteroaralkyl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkyl, an optionally substituted nucleoside, an optionallysubstituted amino acid, an optionally substituted di-, tri- ortetra-peptide, —CH₂—C(O)—C(O)—O—R′ or —CH═C(OH)—C(O)—O—R′; X is:—N(R′)—, —S—, —S(O)—, —S(O)₂—, —S—Y—S—, or a covalent bond to the sulfuratom of Cys or to the nitrogen atom of optionally substitutedheterocyclyl; Y is: optionally substituted aryl, optionally substitutedheteroaryl, an optionally substituted nucleoside, an optionallysubstituted amino acid, or an optionally substituted di-, tri- ortetra-peptide; W is: ═O, ═N—OR^(a), ═N—NR^(b)R^(c), or —N(OH)—R^(d); Zis: —OR, —SR, or —NR^(b)R^(c); R′ is: independently selected fromhydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl or optionally substituted aryl; R is: hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, optionally substituted heteroaralkyl, optionally substitutedheterocyclyl or optionally substituted heterocycloalkyl; R^(a) is:hydrogen, optionally substituted alkyl, optionally substituted aryl,optionally substituted aralkyl, or alkenyl; R^(b) is: independentlyselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl or optionallysubstituted cycloalkyl; R^(c) is: independently selected from hydrogenor optionally substituted alkyl; and R^(d) is: hydrogen, acyl oroptionally substituted alkyl; or R^(b) and R^(c) together with thenitrogen to which they are attached may form a 5- to 7-membered ring,optionally incorporating one or two additional ring heteroatoms chosenfrom N, S, or O, and said ring being optionally substituted with one ormore substituents independently selected from the group consisting of═O, =S, acyl, optionally substituted alkenyl, optionally substitutedalkyl, (optionally substituted alkoxy)carbonyl, and (optionallysubstituted amino) carbonyl; including single tautomers, singlestereoisomers and mixtures of tautomers and/or stereoisomers, and thepharmaceutically acceptable salts thereof, provided that where X is —S—,W is ═O, and Z is OH, A is not 6-amino-3,5-dicyano-4-(optionallysubstituted phenyl)-pyridin-2-yl.
 2. The method of claim 1, wherein: Ais: optionally substituted alkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted heteroaryl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkyl, an optionally substituted amino acid, or an optionallysubstituted di-, tri- or tetra-peptide; X is: —N(H)—, —S—, or a covalentbond to the sulfur atom of Cys or to the nitrogen atom of optionallysubstituted heterocyclyl; W is: ═O or ═N—OR^(a); Z is: —OR, or—NR^(b)R^(c); R is: hydrogen, optionally substituted alkyl, substitutedcycloalkyl, or optionally substituted aralkyl; R^(a) is: hydrogen,optionally substituted alkyl, optionally substituted aryl, or optionallysubstituted aralkyl; R^(b) is: hydrogen, optionally substituted alkyl,optionally substituted aryl, optionally substituted aralkyl oroptionally substituted cycloalkyl; R^(c) is: hydrogen or optionallysubstituted alkyl; and R^(b) and R^(c) together with the nitrogen towhich they are attached may form a 5- or 6-membered ring, optionallyincorporating N or O as an additional ring heteroatom, and said ringbeing optionally substituted with one substituent selected from thegroup consisting of acyl and optionally substituted alkyl.
 3. The methodof claim 2, wherein: A is: an optionally substituted amino acid selectedfrom Ala, Asp, Cys, Glu and Gly, or an optionally substituted di- ortri-peptide the amino acids of which are selected from Ala, Asp, Cys,Glu and Gly;
 4. The method of claim 3, wherein: A is: the tri-peptideGlu-Cys-Gly; and X is: a covalent bond to the sulfur atom of Cys.
 5. Themethod of claim 4, wherein: R is: hydrogen or C₁ to C₈ alkyl; R^(a) is:hydrogen, C₁ to C₈ alkyl or alkenyl, phenyl or aralkyl; R^(b) is C₁ toC₈ optionally acyl-substituted alkyl, optionally substituted aralkyl orcycloalkyl; and R^(c) is: hydrogen or C₁ to C₄ alkyl; or R^(b) and R^(c)together with the nitrogen to which they are attached form a 5-memberedring, or a 6-membered ring optionally incorporating O as an additionalring heteroatom, and said ring being optionally substituted with onesubstituent selected from the group consisting of acyl and optionallysubstituted alkyl.
 6. The method of claim 5, wherein the compound ofFormula I is selected from:2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-oxo-2-pentyloxycarbonyl-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexyloxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]butyricacid,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]butyricacid,2-amino-4-[2-(2-butoxycarbonyl-2-methoxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,2-amino-4-[2-(2-benzyloxyimino-2-butoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,2-amino-4-[2-(2-butoxycarbonyl-2-hydroxyimino-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid2-amino-4-[2-(2-benzyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the HCl slat thereof,2-amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-ethoxycarbonyl-2-(4-nitro-benzyoxyimino)-ethylsulfanyl]-ethylcarbamoyl}-butyricacid,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-phenoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid or the HCl salt thereof,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-ethoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid or the di-HCl salt thereof,2-amino-4-[2-(2-tert-butoxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid or the di-HCl salt thereof,4-[2-(2-allyloxyimino-2-ethoxycarbonyl-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-2-amino-butyric acid or thedi-HCl salt thereof,2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[3-(4-methyl-piperidin-1-yl)-2,3-dioxo-propylsulfanyl]-ethylcarbamoyl}-butyricacid,2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hydroxyimino-3-oxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-diethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-piperidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[2-(1-methoxycarbonyl-2-phenyl-ethylcarbamoyl)-2-oxo-ethylsulfanyl]-ethylcarbamoyl}-butyricacid,2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-cyclohexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,2-Amino-4-[2-(2-benzylcarbamoyl-2-oxo-ethylsulfanyl)-1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyricacid,2-Amino-4-[1-(carboxymethyl-carbamoyl)-2-(3-morpholin-4-yl-2,3-dioxo-propylsulfanyl)-ethylcarbamoyl]-butyricacid,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-ethoxycarbonyl-2-methoxyimino-ethylsulfanyl)-ethylcarbamoyl]-butyricacid2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2,3-dioxo-3-pyrrolidin-1-yl-propylsulfanyl)-ethylcarbamoyl]-butyricacid,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-octylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,1-{3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionyl}-pyrrolidine-2-carboxylicacid methyl ester,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-hexylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid,2-{3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-oxo-propionylamino}-3-methyl-pentanoicacid methyl ester,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-dimethylcarbamoyl-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid, and2-amino-4-(1-(carboxymethyl-carbamoyl)-2-{2-[2-(4-hydroxy-phenyl)-1-methoxycarbonyl-ethylcarbamoyl]-2-oxo-ethylsulfanyl}-ethylcarbamoyl)-butyricacid.
 7. The method of claim 2, wherein: A is: substituted alkyl,substituted aryl, optionally substituted heteroaryl, heterocyclyl, orsubstituted heterocycloalkyl.
 8. The method of claim 7, wherein: R is:hydrogen or C₁ to C₈ alkyl; R^(a) is: hydrogen, C₁ to C₈ alkyl, oraralkyl; R^(b) is: C₁ to C₄ alkyl, optionally substituted aralkyl orcycloalkyl; and R^(c) is: hydrogen or C₁ to C₄ alkyl; or R^(B) and R^(c)together with the nitrogen to which they are attached form a 6-memberedring, optionally incorporating o as an additional ring heteroatom, andsaid ring being optionally substituted with one substituent selectedfrom the group consisting of acyl and optionally substituted alkyl. 9.The method of claim 8, wherein the compound of Formula I is selectedfrom: 3-(1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acid,2-oxo-3-(4-oxo-3,4-dihydro-quinazolin-2-ylsulfanyl)-propionic acid ethylester, 3-[1-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacid ethyl ester, 3-(benzoselenazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester, 3-(1H-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester, 3-(5-chloro-benzothiazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester, 3-(5-nitro-benzoimidazol-2-ylsulfanyl)-2-oxo-propionic acidethyl ester, 3-(5-methoxy-benzoimidazol-2-ylsulfanyl)-2-oxo-propionicacid ethyl ester, 3-(4,5-dihydro-thiazol-2-ylsulfanyl)-2-oxo-propionicacid ethyl ester, 2-hydroxyimino-3-p-tolylsulfanyl-propionic acid methylester, 2-hydroxyimino-3-p-tolylsulfanyl-propionic acid,2-hydroxyimino-3-p-tolylsulfanyl-propionic acid ethyl ester,3-(5-chloro-benzothiazol-2-ylsulfanyl)-2-hydroxyimino-propionic acidethyl ester,2-hydroxyimino-3-(5-methoxy-1H-benzoimidazol-2-ylsulfanyl)-propionicacid ethyl ester,3-(1H-benzoimidazol-2-ylsulfanyl)-2-hydroxyimino-propionic acid ethylester, 2-hydroxyimino-N-phenyl-3-p-tolylsulfanyl-propionamide, and1-piperidin-1-yl-3-p-tolylsulfanyl-propane-1,2-dione 2-oxime.
 10. Themethod of claim 1 wherein the condition characterized by oxidativestress is selected from: ischemia including stroke, cerebral ischemia,retinal ischemia, myocardial ischemia, myocardial infarction andpost-surgical cognitive dysfunction; neurodegenerative disordersincluding Alzheimer's, dementia and Parkinson's disease; peripheralneuropathy, including spinal cord injury, head injury and surgicaltrauma; inflammatory disorders including diabetes, renal disease,pre-menstrual syndrome, asthma, cardiopulmonary inflammatory disorders,heart failure, rheumatoid arthritis, osteoarthritis, muscle fatigue andintermittent claudication; and for the preservation of allograft tissueand organs for transplantation.
 11. The method of claim 1 wherein thecondition characterized by oxidative stress is selected from: myocardialischemia, myocardial infarction, cardiopulmonary inflammatory disorders;heart failure.
 12. The method of claim 11 wherein: W is: ═O; and Z is:—OR.
 13. The method of claim 1 wherein the condition characterized byoxidative stress is selected from: stroke, cerebral ischemia, retinalischemia, peripheral neuropathy including spinal cord injury, headinjury and surgical trauma, and neurodegenerative disorders includingAlzheimer's, dementia and Parkinson's disease.
 14. The method of claim13 wherein: W is: ═o or ═N—OR^(a); and Z is: —OR, or —NR^(b)R^(c). 15.The method of claim 14 wherein: W is: ═N—OR^(a); and Z is: —NR^(b)R^(c).16. The method of claim 1 wherein the condition characterized byoxidative stress is selected from: diabetes; renal disease;pre-menstrual syndrome; asthma, rheumatoid arthritis; muscle fatigue;and intermittent claudication; and for the preservation of allografttissue and organs for transplantation.
 17. A method of treating a mammalhaving a condition characterized by oxidative stress, comprisingadministering a therapeutically effective amount of a compound ofFormula II:

wherein: R¹ is: hydrogen, optionally substituted alkyl, optionallysubstituted aryl, —C(O)—O—R′, —CH₂—SH, —CH₂—S—CH₂—C(W)—C(O)—Z,—CH₂—S—CH═C(OH)—C(O)—Z, —CH₂—S(O)—CH₂—C(W)—C(O)—Z, or—CH₂—S(O)—CH═C(OH)—C(O)—Z′; R² is: hydrogen, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted acyl; R³ is: independently selected fromhydrogen, optionally substituted lower alkyl, optionally substitutedaralkyl, —CH₂—SH, —CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z; R⁴ is:hydrogen, optionally substituted lower alkyl, optionally substitutedaralkyl, optionally substituted heteroararalkyl, —CH₂—SH,—CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z; R⁵ is:hydrogen, optionally substituted alkyl, or optionally substituted aryl;R′ is: independently selected from hydrogen, optionally substitutedalkyl, or optionally substituted aryl; W is: ═O, ═N—OR^(a),═N—NR^(b)R^(c); or —N(OH)—R^(d) Z is: —OR, —SR, or —NR^(b)R^(c); R is:hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl, optionally substituted heteroaralkyl,optionally substituted heterocyclyl or optionally substitutedheterocycloalkyl; R^(a) is: hydrogen, optionally substituted alkyl,optionally substituted aryl, optionally substituted aralkyl, or alkenyl;R^(b) is: independently selected from hydrogen, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl oroptionally substituted cycloalkyl, R^(c) is: independently selected fromhydrogen or optionally substituted alkyl; and R^(d) is: hydrogen, acylor optionally substituted alkyl; or R^(b) and R^(c) together with thenitrogen to which they are attached may form an 5- to 7-membered ring,optionally incorporating one or two additional ring heteroatoms chosenfrom N, S, or O, and said ring being optionally substituted with one ormore substituents independently selected from the group consisting of═O, ═S, acyl, optionally substituted alkenyl, optionally substitutedalkyl, (optionally substituted alkoxy)carbonyl, and (optionallysubstituted amino)carbonyl; k is: 0, 1 or 2; m is: 0, 1 or 2; and n is:0, 1, 2 or 3; including single tautomers, single stereoisomers andmixtures of tautomers and/or stereoisomers, and the pharmaceuticallyacceptable salts thereof, provided that at least one of R¹, R³ or R⁴ is—CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z.
 18. The methodof claim 17, wherein: R¹ is: —C(O)—O—R′ where R′ is hydrogen, or—CH₂—S—CH₂—C(O)—C(O)—O—R′ or —CH₂—S—CH═C(OH)—C(O)—O—R′ where R′ ishydrogen or lower alkyl; R² is: hydrogen; R³ is: —CH₂—SH,—CH₂—S—CH₂—C(W)—C(O)—Z, —CH₂—S—CH═C(OH)—C(O)—Z,—CH₂—S(O)—CH₂—C(W)—C(O)—Z, or —CH₂—S(O)—CH═C(OH)—C(O)—Z R⁴ is: hydrogen;R⁵ is hydrogen or lower alkyl; W is: ═O or ═N—OR^(a); Z is: —OR or—NR^(b)R^(c); R is: hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, or optionallysubstituted aralkyl; R^(a) is: hydrogen or alkyl; R^(b) is: C₁ to C₄alkyl, phenyl or benzyl; R^(c) is: hydrogen or C₁ to C₄ alkyl, or R^(b)and R^(c) together with the nitrogen to which they are attachwed form a6-membered ring selected from 4-optionally substituted-piperidin-1-yland morpholin-4-yl; and k, m and n are respectively: 0,2,1; 1,0,1; or2,0,1.
 19. The method of claim 18, wherein: R¹ is: —COOH; R⁵ is:hydrogen; and k, m and n are respectively: 0,2,1; or 2,0,1.
 20. Themethod of claim 17 wherein the condition characterized by oxidativestress is selected from: ischemia including stroke, cerebral ischemia,retinal ischemia, myocardial ischemia, myocardial infarction andpost-surgical cognitive dysfunction; neurodegenerative disordersincluding Alzheimer's, dementia and Parkinson's disease; peripheralneuropathy, including spinal cord injury, head injury and surgicaltrauma; inflammatory disorders including diabetes, renal disease,pre-menstrual syndrome, asthma, cardiopulmonary inflammatory disorders,heart failure, rheumatoid arthritis, osteoarthritis, muscle fatigue andintermittent claudication; and for the preservation of allograft tissueand organs for transplantation
 21. The method of claim 19 wherein thecondition characterized by oxidative stress is selected from: ischemiaincluding stroke, cerebral ischemia, retinal ischemia, myocardialischemia, myocardial infarction and post-surgical cognitive dysfunction;neurodegenerative disorders including Alzheimer's, dementia andParkinson's disease; peripheral neuropathy, including spinal cordinjury, head injury and surgical trauma; inflammatory disordersincluding diabetes, renal disease, pre-menstrual syndrome, asthma,cardiopulmonary inflammatory disorders, heart failure, rheumatoidarthritis, osteoarthritis, muscle fatigue and intermittent claudication;and for the preservation of allograft tissue and organs fortransplantation.
 22. A method of treating a mammal having a conditioncharacterized by oxidative stress, comprising administering atherapeutically effective amount of a compound of Formula III:

wherein: R^(3.1) is: H or is absent; R^(3.2) is: H or C₁ to C₄ alkyl;R^(3.3) and R^(3.4) are both H or are both C₁ to C₄ alkyl; and R^(3.5)is: H, COOH, or —C(O)O—(C₁ to C₄ alkyl); including single tautomers,single stereoisomers and mixtures of tautomers and/or stereoisomers, andthe pharmaceutically acceptable salts thereof.
 23. The method of claim22 wherein: R^(3.2) is: H or ethyl; R^(3.3) and R^(3.4) are both H orare both methyl; and R^(3.5) is: COOH;
 24. The method of claim 22,wherein: R^(3.3) and R^(3.4) are both H or are both methyl; and R^(3.5)is: COOH.
 25. The method of claim 24, wherein: R^(3.1) is hydrogen; andR^(3.2) is hydrogen or ethyl.
 26. The method of claim 22 wherein thecondition characterized by oxidative stress is selected from: ischemiaincluding stroke, cerebral ischemia, retinal ischemia, myocardialischemia, myocardial infarction and post-surgical cognitive dysfunction;neurodegenerative disorders including Alzheimer's, dementia andParkinson's disease; peripheral neuropathy, including spinal cordinjury, head injury and surgical trauma; inflammatory disordersincluding diabetes, renal disease, pre-menstrual syndrome, asthma,cardiopulmonary inflammatory disorders, heart failure, rheumatoidarthritis, osteoarthritis, muscle fatigue and intermittent claudication;and for the preservation of allograft tissue and organs fortransplantation.